WO2025106930A1 - Polyribonucléotides circulaires codant le peptide 2 de type glucagon (glp-2) et leurs utilisations - Google Patents

Polyribonucléotides circulaires codant le peptide 2 de type glucagon (glp-2) et leurs utilisations Download PDF

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WO2025106930A1
WO2025106930A1 PCT/US2024/056294 US2024056294W WO2025106930A1 WO 2025106930 A1 WO2025106930 A1 WO 2025106930A1 US 2024056294 W US2024056294 W US 2024056294W WO 2025106930 A1 WO2025106930 A1 WO 2025106930A1
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seq
circular polyribonucleotide
sequence
polypeptide
domain
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Russell Miller
John Dwyer
Ewen Cameron
Yashu Liu
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Sail Biomedicines Inc
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Sail Biomedicines Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • Glucagon-like peptide (GLP)-2 is a peptide hormone derived from tissue-specific posttranslational processing of the pro-glucagon peptide.
  • GLP-2 and GLP-2 analogs have been used to treat intestinal diseases, such as short bowel syndrome.
  • the present disclosure is based, at least in part, on the development of circular polyribonucleotides and modified RNAs encoding a GLP-2 polypeptide or analog thereof. These circular polyribonucleotides and modified RNAs provide for prolonged and stable expression of the GLP-2 polypeptide or analog thereof in a subject (e.g., subjects having a GLP- 2-related disease, e.g., intestinal diseases such as short bowel syndrome).
  • circular polyribonucleotides including a sequence encoding a polypeptide including (i) a glucagon-like peptide-2 (GLP-2) polypeptide or analog thereof, (ii) a half-life extension moiety, and (iii) a secretion signal sequence.
  • GLP-2 polypeptide or analog thereof includes an amino acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.
  • the GLP-2 polypeptide or analog thereof includes an amino acid sequence of any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.
  • the sequence encoding the polypeptide includes a nucleic acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.
  • the sequence encoding the polypeptide includes a nucleic acid sequence of any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.
  • the GLP-2 analog includes teduglutide, glepaglutide, dapiglutide, apraglutide, HM15912, and SHP-681.
  • the half-life extension moiety includes a peptide moiety.
  • the peptide moiety includes an Fc domain, an albumin polypeptide, a nanobody, or a post-translational modification site.
  • the Fc domain includes an IgG1 Fc domain, an IgG2 Fc domain, an IgG3 Fc domain, or an IgG4 Fc domain.
  • the Fc domain includes an amino acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identical to SEQ ID NO: 15 (IgG1 Fc domain), SEQ ID NO: 17 (IgG2 Fc domain), SEQ ID NO: 19 (IgG3 Fc domain), or SEQ ID NO: 21 (IgG4 Fc domain).
  • the Fc domain includes an amino acid sequence of SEQ ID NO: 15 (IgG1 Fc domain), SEQ ID NO: 17 (IgG2 Fc domain), SEQ ID NO: 19 (IgG3 Fc domain), or SEQ ID NO: 21 (IgG4 Fc domain).
  • the IgG4 Fc domain includes an amino acid sequence of SEQ ID NO: 21.
  • the sequence encoding the polypeptide includes a nucleic acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16 (IgG1 Fc domain), SEQ ID NO: 18 (IgG2 Fc domain), SEQ ID NO: 20 (IgG3 Fc LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 domain), or SEQ ID NO: 22 (IgG4 Fc domain).
  • the sequence encoding the polypeptide includes a nucleic acid sequence of SEQ ID NO: 16 (IgG1 Fc domain), SEQ ID NO: 18 (IgG2 Fc domain), SEQ ID NO: 20 (IgG3 Fc domain), or SEQ ID NO: 22 (IgG4 Fc domain).
  • the Fc domain includes a mutation.
  • the Fc domain includes the amino acid sequence of SEQ ID NO: 15 (IgG1 Fc domain), SEQ ID NO: 17 (IgG2 Fc domain), SEQ ID NO: 19 (IgG3 Fc domain), or SEQ ID NO: 21 (IgG4 Fc domain), except the amino acid sequence of SEQ ID NO: 15 (IgG1 Fc domain), SEQ ID NO: 17 (IgG2 Fc domain), SEQ ID NO: 19 (IgG3 Fc domain), or SEQ ID NO: 21 (IgG4 Fc domain) includes an amino acid substitution selected from one or more of: M252Y/S254T/T256E (YTE); L284A/L285A; M428L/N434S (LS); T307A/E380A/N434A (AAA); T250Q/M428L (QL), and V308P.
  • the albumin polypeptide includes an amino acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23.
  • the peptide moiety includes a nanobody that binds to albumin.
  • the circular polyribonucleotide includes one or more modifications.
  • the one or more modifications include one or more modifications to a portion of the sequence encoding the GLP-2 polypeptide or analog thereof.
  • the one or more modifications include a modification to a sugar, a nucleobase, an internucleoside linkage, or a combination thereof.
  • the circular polyribonucleotide includes a translation initiation sequence operably linked to the sequence encoding the polypeptide. In some embodiments, the circular polyribonucleotide includes an internal ribosome entry site (IRES) operatively linked to the sequence encoding the polypeptide. In some embodiments, the circular polyribonucleotide includes a translation termination sequence. In some embodiments, the circular polyribonucleotide lacks a translation termination sequence. In some embodiments, the circular polyribonucleotide includes a stagger element at a 3’ end of the sequence encoding the polypeptide. In some embodiments, the stagger element is configured to stall a ribosome during rolling circle translation.
  • the circular polyribonucleotide includes a replication element. LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1
  • the circular polyribonucleotide includes a 5’ untranslated region operably linked to the sequence encoding the polypeptide.
  • the circular polyribonucleotide does not include a poly(A) sequence operably linked to the sequence encoding the polypeptide.
  • the circular polyribonucleotide does not include a 5’ untranslated region operably linked to the sequence encoding the polypeptide.
  • the secretion signal sequence is a liver-specific secretion signal sequence, where the liver-specific secretion signal includes an amino acid sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87
  • the secretion signal sequence includes an IL-2 secretion signal sequence, a proglucagon secretion signal sequence, a Gaussia luciferase secretion signal sequence, or a erythropoietin (EPO) secretion signal sequence.
  • the IL-2 secretion signal sequence includes an amino acid sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 29.
  • the proglucagon secretion signal sequence includes an amino acid sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 31.
  • the Gaussia luciferase secretion signal sequence includes an amino acid sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 33.
  • the EPO secretion signal sequence includes an amino acid sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 35. LRN Ref. No.: LRN23-107WO Fish Ref.
  • the sequence encoding the polypeptide includes an amino acid sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to one of SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39.
  • the circular polyribonucleotide includes at least one spacer sequence.
  • the polypeptide includes a protease cleavage site and the at least one spacer sequence encodes the protease cleavage site.
  • the protease cleavage site is positioned between (a) the secretion signal and (b) the GLP-2 polypeptide or analog thereof and the half-life extension moiety.
  • the polypeptide includes at least one linker sequence.
  • the at least one linker sequence encodes an amino acid sequence selected from (GS)x, (GGS)x, (GGGGS)x (SEQ ID NO: 141), (GGSG)x (SEQ ID NO: 142), and (SGGG)x (SEQ ID NO: 143), where x is an integer from 1 to 50.
  • the at least one linker sequence is disposed between the GLP-2 polypeptide or analog thereof and the half-life extension moiety.
  • the circular polyribonucleotide encodes an amino acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the circular polyribonucleotide includes an amino acid sequence of SEQ ID NO: 1. In some embodiments, the circular polyribonucleotide includes a nucleic acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. In some embodiments, the circular polyribonucleotide includes a nucleic acid sequence of SEQ ID NO: 2.
  • compositions including any of the circular polyribonucleotides described herein and a pharmaceutically acceptable excipient.
  • the circular polyribonucleotide is formulated as a lipid nanoparticle.
  • the circular polyribonucleotide is formulated for unencapsulated delivery or natural particle delivery.
  • the pharmaceutical composition is formulated for intravenous, subcutaneous, intrahepatic, or intramuscular administration.
  • the pharmaceutical composition is formulated for local administration.
  • the pharmaceutical composition is formulated for systemic administration. LRN Ref. No.: LRN23-107WO Fish Ref.
  • the pharmaceutical composition includes one or more of the following features: (i) a concentration of the circular polyribonucleotide of between 0.1 ng/mL and 500 mg/mL; (ii) a concentration of deoxyribonucleotide of no more than 100 ng/mL; (iii) a concentration of protein of less than 100 ng of protein per milligram (mg) of the circular polyribonucleotide; (iv) an A260/A280 absorbance ratio of from about 1.6 to 2.3 as measured by a spectrophotometer; (v) substantially free of a process-related impurity selected from a cell protein, a cell deoxyribonucleic acid, an enzyme, a reagent component, a gel component, or a chromatographic material; and (vi) a reduced level of one or more markers of an immune or inflammatory response after purification compared to prior to purification.
  • GLP-2-related disease is selected from the group consisting of: short bowel syndrome (SBS), short bowel syndrome with intestinal failure (SBS-IF), pediatric SBS, adult SBS, major small bowel resection, total parenteral nutrition (TPN)-induced intestinal hypoplasia, small and large intestinal inflammation, Crohn’s disease, chemotherapy- induced mucositis, and intestinal ischemia–reperfusion injury.
  • SBS short bowel syndrome
  • SBS-IF short bowel syndrome with intestinal failure
  • TPN total parenteral nutrition
  • TPN total parenteral nutrition
  • increasing intestinal growth includes increasing crypt cell growth.
  • methods of increasing intestinal absorption of fluids and nutrients in a subject in need thereof including administering to the subject an effective amount of any of the circular polyribonucleotides described herein or any of the pharmaceutical compositions described herein.
  • the subject has short bowel syndrome (SBS), short bowel syndrome with intestinal failure (SBS-IF), pediatric SBS, adult SBS, major small bowel resection, total parenteral nutrition (TPN)-induced intestinal hypoplasia, small and large LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 intestinal inflammation, Crohn’s disease, chemotherapy-induced mucositis, and intestinal ischemia–reperfusion injury.
  • any of the circular polyribonucleotides described herein or any of the pharmaceutical compositions described herein is administered one or more times to the subject.
  • any of the circular polyribonucleotides described herein or any of the pharmaceutical compositions described herein is administered locally. In some embodiments, any of the circular polyribonucleotides described herein or any of the pharmaceutical compositions described herein is administered systemically. In some embodiments, the method includes administering one or more additional therapeutic agents to the subject. In some embodiments, the subject is a human subject. Also provided herein are kits including: (a) (i) a composition including any of the circular polyribonucleotides described herein or (ii) any of the pharmaceutical compositions described herein; and (b) instructions for administering the composition or the pharmaceutical composition to a subject in need thereof.
  • circular polyribonucleotides including a sequence encoding a polypeptide including (i) a glucagon-like peptide-2 (GLP-2) polypeptide or analog thereof, and (ii) a half-life extension moiety. Also provided herein are circular polyribonucleotides including a sequence encoding a polypeptide including (i) a glucagon-like peptide-2 (GLP-2) polypeptide or analog thereof, and (ii) a half-life extension moiety, where the half-life extension moiety is an Fc domain including a M252Y/S254T/T256E (YTE) mutation.
  • GLP-2 glucagon-like peptide-2
  • YTE M252Y/S254T/T256E
  • modified linear RNAs including a sequence encoding a polypeptide including a glucagon-like peptide-2 (GLP-2) or analog thereof. Also provided herein are modified linear RNAs including a sequence encoding a polypeptide including (i) a glucagon-like peptide-2 (GLP-2) polypeptide or analog thereof, and (ii) a secretion signal sequence. In some embodiments, the modified linear RNA further includes a half-life extension moiety (e.g., any of the half-life extension moieties described herein). All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, LRN Ref.
  • each when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection, unless expressly stated otherwise, or unless the context of the usage clearly indicates otherwise.
  • all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
  • the materials, methods, and examples are illustrative only and not intended to be limiting.
  • FIG. 1 includes a graph showing activity of teduglutide in cells treated with circular RNA encoding teduglutide-Fc or recombinant teduglutide.
  • FIGs. 1 includes a graph showing activity of teduglutide in cells treated with circular RNA encoding teduglutide-Fc or recombinant teduglutide.
  • FIG. 2A-2B include graphs showing expression (FIG. 2A) and activity (FIG. 2B) of teduglutide-Fc in mammalian cells transfected with circular RNA constructs encoding teduglutide-Fc.
  • FIG. 3 includes a graph showing levels of teduglutide-Fc in mice injected with circular RNA encoding teduglutide-Fc and reported levels of glepaglutide, teduglutide, apraglutide, and HM15912 in rats injected with recombinant glepaglutide, teduglutide, apraglutide, or HM15912.
  • FIGs. 4A-4F include data from characterization of various LNP formulations including expression levels of teduglutide-Fc (FIG. 4A), complete blood cell count (CBC) (FIGs. 4B-4C), ALT levels (FIG. 4D), IFN ⁇ levels (FIG. 4E), and cytokine levels (FIG. 4F).
  • FIGs. 5A-5G include dose response data in mice injected with LNP formulations of circular RNA encoding teduglutide-Fc. The data include expression of GLP2-Fc (FIGs. 5A-B), cytokine levels (FIG. 5C-F), and anti-target antibody(ATA) response (FIG. 5G) FIGs.
  • FIG. 6A-F include data from characterization of GLP-2 Fc expressed from circular RNA in non-human primates including Teduglutide-Fc expression (FIG. 6A), antibody production (FIG. 6B), complete blood count, including lymphocyte count (FIG. 6C), cytokine responses (FIG. 6D), CRP levels (FIG. 6E), and activate partial thromboplastin (FIG. 6F).
  • FIG. 6A Teduglutide-Fc expression
  • FIG. 6B antibody production
  • FIG. 6C complete blood count, including lymphocyte count (FIG. 6C), cytokine responses (FIG. 6D), CRP levels (FIG. 6E), and activate partial thromboplastin (FIG. 6F).
  • DETAILED DESCRIPTION Provided herein are circular polyribonucleotides and modified RNAs encoding a GLP-2 polypeptide or analog thereof and related pharmaceutical compositions and kits.
  • Circular polyribonucleotides and modified RNAs described herein comprise a sequence encoding a polypeptide comprising (i) a GLP-2 polypeptide or analog thereof, (ii) a half-life extension moiety, and optionally, (iii) a secretion signal sequence.
  • the circular polyribonucleotides and modified RNAs comprise a sequence encoding a polypeptide comprising (i) a GLP-2 polypeptide or analog thereof and (ii) a half-life extension moiety.
  • the circular polynucleotides and modified RNAs comprise a sequence encoding a polypeptide comprising (i) a GLP-2 polypeptide or analog thereof, (ii) a half-life extension moiety, and (iii) a secretion signal sequence.
  • the circular polyribonucleotides and modified RNAs described herein are useful in the treatment or prevention of a GLP-2-related disease (e.g., short bowel syndrome).
  • Circular Polyribonucleotides/Modified RNAs comprise a sequence encoding a polypeptide comprising: (i) a GLP-2 polypeptide or analog thereof, (ii) a half-life extension moiety, and optionally, (iii) a secretion signal sequence, each of which is described herein.
  • a circular polyribonucleotide or a modified RNA comprises a sequence encoding a polypeptide comprising (i) a GLP-2 polypeptide or analog thereof and (ii) a half-life extension moiety.
  • a circular polyribonucleotide or a modified RNA comprise a sequence encoding a polypeptide comprising a sequence encoding a (i) a GLP- 2 polypeptide or analog thereof, (ii) a half-life extension moiety, and (iii) a secretion signal sequence.
  • a circular polyribonucleotide or modified RNA can include two or more copies of the sequence encoding the polypeptide (e.g., any of the exemplary polypeptides described herein).
  • Non-limiting additional sequences that can be included in a circular polyribonucleotide and a modified RNA are described herein.
  • modified RNA or a “linear modified RNA” refers to an RNA molecule that includes one or more (e.g., 2, 3, 4, 5, 10 or more) modified ribonucleotides described herein or modifications any of the exemplary modifications known in the art.
  • modified ribonucleotides include 5-methylcytidine, N1- methylpseudouridine, 5-methoxyuridine, and pseudouridine.
  • a non-limiting example of a sequence encoding a polypeptide that can be present in any of the circular polyribonucleotides or modified RNAs described herein are provided in Table 1 (SEQ ID NO: 2).
  • a non-limiting example of a polypeptide comprising (i) a GLP-2 polypeptide or analog thereof, (ii) a half-life extension moiety, and (iii) a secretion signal sequence is provided in Table 1 (SEQ ID NO: 1). Table 1.
  • SEQ ID NO: 1 SEQ ID NO: 1
  • Table 1 Non-limiting example of a polypeptide and a sequence encoding a polypeptide SEQ Description Sequence ID NO: G T K P A LRN Ref. No.: LRN23-107WO Fish Ref.
  • the polypeptide comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO: 1. In some embodiments, the sequence (encoding the polypeptide) comprises a sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. In some embodiments, the sequence (encoding the polypeptide) comprises a sequence of SEQ ID NO: 2.
  • GLP-2 GLP-2 and Analogs Thereof Pro-glucagon is cleaved to glucagon by prohormone convertase 2 (PC2) in pancreatic ⁇ - cells (and other organs), and it is cleaved to glucagon-like peptide-2 (GLP-2) by prohormone convertase 1 (PC1).
  • GLP-2 is a 33 amino acid peptide-encoded carboxyterminal to the sequence of GLP-1 in the proglucagon gene.
  • GLP-2 also significantly enhances the surface area of the mucosal epithelium via stimulation of crypt cell proliferation and inhibition of apoptosis in the enterocyte and crypt compartments.
  • GLP-1 and GLP-2 are secreted from gut endocrine cells and promote nutrient absorption through distinct mechanisms of action. GLP-2 is also known to regulate gastric motility, gastric acid secretion, intestinal hexose transport, and increases the barrier function of the gut epithelium. GLP-2 has been shown to reduce mortality and decrease mucosal injury, cytokine expression, and bacterial septicemia in LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 the setting of small and large bowel inflammation. GLP-2 also enhances nutrient absorption and gut adaptation in rodents or humans with short bowel syndrome.
  • GLP-2 analog refers to a polypeptide having one or more alterations (e.g., substitutions, deletions, and/or insertions) in an amino acid sequence of wild-type GLP-2 and having at least partial GLP-2 activity as compared to a wild-type GLP-2 polypeptide.
  • Non- limiting examples of GLP-2 analogs include teduglutide, glepaglutide, dapiglutide, apraglutide, HM15912, and SHP-681. Further non-limiting examples of GLP-2 analogs are described in WO 2020/020904, which is incorporated herein by reference in its entirety. Non-limiting examples of amino acid sequences of pro-glucagon, GLP-2, and GLP-2 analogs are shown in Table 2 below. Non-limiting examples of nucleic acid sequence encoding pro-glucagon, GLP-2, and GLP-2 analogs are also shown in Table 2 below. Table 2.
  • the GLP-2 polypeptide or analog thereof comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.
  • the GLP-2 polypeptide or analog thereof comprises an amino acid sequence of any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.
  • the sequence (encoding the GLP-2 polypeptide or analog thereof) comprises a sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.
  • the sequence (encoding the GLP-2 polypeptide or analog thereof) comprises a sequence of any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.
  • the GLP-2 polypeptide or analog thereof comprises an amino acid sequence that is a functional fragment of any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13. LRN Ref. No.: LRN23-107WO Fish Ref.
  • the GLP-2 polypeptide or analog thereof comprises a functional fragment that has a sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.
  • the GLP-2 polypeptide or analog thereof comprises an amino acid sequence of SEQ ID NO: 13.
  • the amino acid at position X 1 is norleucine.
  • the amino acid at position X 2 is D-phenylalanine.
  • GLP-2 polypeptide or analog thereof comprises an amino acid sequence of SEQ ID NO: 167.
  • the amino acid at positions X1, X2, X3, and X 4 of SEQ ID NO: 167 can be any amino acid.
  • each of X1, X2, X3, and X4 can independently be any of amino acids alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine, with the proviso that the combination of X 1 of histidine, X 2 of alanine, X 3 of aspartic acid, and X4 of glycine is excluded.
  • a “functional fragment” is a fragment that retains at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the activity of the wildtype polypeptide from which it is derived.
  • a functional fragment can have an activity greater than the wildtype polypeptide from which it is derived.
  • the GLP-2 polypeptide or analog thereof comprises an amino acid sequence having at least one mutation.
  • substitution refers to a substitution of a residue within a sequence (e.g., a nucleic acid or an amino acid sequence) with another residue, or a deletion or insertion of one or more residues within a sequence (e.g., a nucleic acid or an amino acid sequence). Substitutions are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions provided herein are well known in the art, and are provided by, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012).
  • the GLP-2 polypeptide or analog thereof can comprise any number and/or any type of substitutions.
  • the sequence encoding the GLP-2 polypeptide or analog thereof comprises one or more mutations as compared to the wild type sequence.
  • the one or more mutations can be silent, i.e., they do not modify the amino acid sequence of any encoded protein (or otherwise result in a substituted amino acid sequence).
  • the one or more mutations in a nucleic acid sequence can result in modifications to the encoded amino acid sequence of GLP-2 or analog thereof, resulting in GLP-2 or analog thereof having one or more amino acid substitutions (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid substitutions) relative to the wild-type protein sequence.
  • the polypeptide comprises (i) a GLP-2 polypeptide or analog thereof (e.g., any of the GLP-2 polypeptides or analogs thereof described herein or known in the art) and (ii) a half-life extension moiety (e.g., any of the exemplary half-life extension moieties described herein or known in the art).
  • the polypeptide comprises (i) a secretion signal sequence (e.g., any of the exemplary secretion signal sequences described herein or known in the art), (ii) a GLP-2 polypeptide or analog thereof (e.g., any of the exemplary GLP- 2 polypeptides or analogs thereof described herein or known in the art), and (iii) a half-life extension moiety (e.g., any of the exemplary half-life extension moieties described herein or known in the art).
  • a secretion signal sequence e.g., any of the exemplary secretion signal sequences described herein or known in the art
  • GLP-2 polypeptide or analog thereof e.g., any of the exemplary GLP- 2 polypeptides or analogs thereof described herein or known in the art
  • a half-life extension moiety e.g., any of the exemplary half-life extension moieties described herein or known in the art.
  • the polypeptide may further comprise an epitope tag for detection or purification (e.g., a HiBiT-tag) and/or a fluorescent tag for visualization (e.g., a GFP-tag).
  • modified linear RNAs including a sequence encoding a polypeptide including a glucagon-like peptide-2 (GLP-2) or analog thereof.
  • modified linear RNAs including a sequence encoding a polypeptide including (i) a glucagon-like peptide-2 (GLP-2) polypeptide or analog thereof, and (ii) a secretion signal sequence.
  • the modified linear RNA includes a half-life extension moiety (e.g., any of the half-life extension moieties described herein).
  • a half-life extension moiety e.g., any of the half-life extension moieties described herein.
  • the polypeptide encoded by a sequence in a circular polyribonucleotide or a modified RNA described herein
  • the term “half-life extension moiety” refers to any moiety that extends the circulating serum LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 half-life of a polypeptide comprising a GLP-2 polypeptide or analog thereof in vivo.
  • Circulating serum half-life refers to the amount of time it takes for the concentration or amount of a polypeptide comprising a GLP-2 polypeptide or analog thereof in the serum to be reduced by half (i.e., 50%).
  • the half-life extension moiety is a peptide moiety.
  • Non-limiting examples of a half-life extension moiety include an antibody or fragment thereof (e.g., an Fc domain, e.g., an IgG1 Fc domain, an IgG2 Fc domain, an IgG3 Fc domain, or an IgG4 Fc domain), a serum protein or fragment thereof (e.g., albumin or fragment thereof (e.g., human serum albumin or a fragment thereof), fibronectin or fragment thereof (e.g., human fibronectin or a fragment thereof), or transferrin or fragment thereof (e.g., human transferrin or a fragment thereof), and antibodies or an antigen-binding fragments thereof or peptides that bind to a serum protein (e.g., an antibody or antigen-binding fragment thereof that binds to human albumin, human fibronectin, or human transferrin).
  • an antibody or fragment thereof e.g., an Fc domain, e.g., an IgG1 Fc domain, an IgG2
  • Antigen-binding fragments of antibodies include, e.g., single-chain antibodies (scFv), single-domain antibodies (also called nanobodies), Fab fragments, Fab ⁇ fragments, and F(ab ⁇ )2 Fab2 fragments, diabodies, triabodies, and tetrabodies.
  • scFv single-chain antibodies
  • nanobodies single-domain antibodies
  • Fab fragments Fab ⁇ fragments
  • F(ab ⁇ )2 Fab2 fragments diabodies, triabodies, and tetrabodies.
  • Non-limiting examples of amino acid sequences (and corresponding nucleic acid sequences) of half-life extension moieties are shown in Table 3 below. Table 3.
  • Non-limiting examples of sequences of half-life extension moieties SEQ Description Sequence ID E L S C C G G C G A C C G T E L LRN Ref. No.: LRN23-107WO Fish Ref.
  • the half-life extension moiety comprises an antibody or fragment thereof.
  • Non-limiting examples of antibody fragments include a Fab, a Fab ⁇ , a F(ab ⁇ )2, a Fc, a Fv, an scFv, a diabody, a triabody, a tetrabody, and a nanobody.
  • the half-life extension moiety comprises an Fc domain (e.g., an IgG1 Fc domain (e.g., a human IgG1 Fc domain), an IgG2 Fc domain (e.g., a human IgG2 Fc domain), an IgG3 Fc domain (e.g., a human IgG3 Fc domain, or an IgG4 Fc domain (e.g., a human IgG4 Fc domain).
  • an Fc domain e.g., an IgG1 Fc domain (e.g., a human IgG1 Fc domain)
  • an IgG2 Fc domain e.g., a human IgG2 Fc domain
  • an IgG3 Fc domain e.g., a human IgG3 Fc domain
  • an IgG4 Fc domain e.g., a human IgG4 Fc domain
  • the half-life extension moiety comprises an Fc domain comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15 (human IgG1 Fc domain), SEQ ID NO: 17 (human IgG2 Fc domain), SEQ ID NO: 19 (human IgG3 Fc domain), or SEQ ID NO: 21 (human IgG4 Fc domain).
  • the half-life extension moiety comprises an Fc domain comprising an amino acid sequence of SEQ ID NO: 15 (human IgG1 Fc domain), SEQ ID NO: 17 (human IgG2 Fc domain), SEQ ID NO: 19 (human IgG3 Fc domain), or SEQ ID NO: 21 (human IgG4 Fc domain).
  • the sequence encoding the Fc domain comprises a nucleic acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID LRN Ref. No.: LRN23-107WO Fish Ref.
  • the sequence encoding the Fc domain comprises a nucleic acid sequence of SEQ ID NO: 16 (human IgG1 Fc domain), SEQ ID NO: 18 (human IgG2 Fc domain), SEQ ID NO: 20 (human IgG3 Fc domain), or SEQ ID NO: 22 (human IgG4 Fc domain).
  • the sequence encoding the Fc domain comprises a nucleic acid sequence of SEQ ID NO: 16 (human IgG1 Fc domain), SEQ ID NO: 18 (human IgG2 Fc domain), SEQ ID NO: 20 (human IgG3 Fc domain), or SEQ ID NO: 22 (human IgG4 Fc domain).
  • the Fc domain comprises an amino acid sequence having at least one substitution.
  • the Fc domain can comprise any number and/or any type of substitution.
  • the Fc domain can comprise any substitution known in the art or described herein.
  • Non-limiting examples of substitutions that can be included in an Fc domain include M252Y/S254T/T256E (YTE); L234A/L235A (LALA); M428L/N434S (LS); T307A/E380A/N434A (AAA); T250Q/M428L (QL); and V308P.
  • the Fc domain can comprise the amino acid sequence of SEQ ID NO: 15 (human IgG1 Fc domain), except that it comprises an amino acid substitution(s) selected from one or more of: M252Y/S254T/T256E (YTE); L234A/L235A (LALA); M428L/N434S (LS); T307A/E380A/N434A (AAA); T250Q/M428L (QL); and V308P.
  • the Fc domain can comprise the amino acid sequence of SEQ ID NO: 17 (human IgG2 Fc domain), except that it comprises an amino acid substitution(s) selected from one or more of: M252Y/S254T/T256E (YTE); L234A/L235A (LALA); M428L/N434S (LS); T307A/E380A/N434A (AAA); T250Q/M428L (QL); and V308P.
  • the Fc domain can comprise the amino acid sequence of SEQ ID NO: 19 (human IgG3 Fc domain), except that it comprises an amino acid substitution(s) selected from one or more of: M252Y/S254T/T256E (YTE); L234A/L235A (LALA); M428L/N434S (LS); T307A/E380A/N434A (AAA); T250Q/M428L (QL); and V308P.
  • the Fc domain can comprise the amino acid sequence of SEQ ID NO: 21 (human IgG4 Fc domain), except that it comprises an amino acid substitution(s) comprising an amino acid substitution selected from one or more of: M252Y/S254T/T256E (YTE); L234A/L235A (LALA); M428L/N434S (LS); T307A/E380A/N434A (AAA); T250Q/M428L (QL); and V308P.
  • the half-life extension moiety comprises an albumin polypeptide or a functional fragment thereof (e.g., a human albumin polypeptide or fragment thereof), a LRN Ref.
  • the albumin polypeptide or a functional fragment thereof comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23. In some embodiments, the albumin polypeptide comprises an amino acid sequence of SEQ ID NO: 23.
  • the albumin polypeptide comprises an amino acid sequence having at least one substitution (e.g., one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions and/or deletions) as compared to the corresponding wildtype albumin polypeptide (e.g., wildtype human albumin polypeptide, e.g., SEQ ID NO: 23).
  • the albumin polypeptide can comprise one, two, three, four, five, six, seven, eight, nine, or ten of any type of substitution.
  • the albumin polypeptide or a functional fragment thereof is encoded by a nucleic acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24. In some embodiments, the albumin polypeptide is encoded by a nucleic acid sequence of SEQ ID NO: 24. In some embodiments, the fibronectin polypeptide or a functional fragment thereof comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 25. In some embodiments, the fibronectin polypeptide comprises an amino acid sequence of SEQ ID NO: 25.
  • the fibronectin polypeptide comprises an amino acid sequence having at least one substitution (e.g., one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions and/or deletions) as compared to the corresponding wildtype fibronectin polypeptide (e.g., wildtype human fibronectin polypeptide, e.g., SEQ ID NO: 25).
  • the fibronectin polypeptide can comprise one, two, three, four, five, six, seven, eight, nine, or ten of any type of substitution.
  • the fibronectin polypeptide or a functional fragment thereof is encoded by a nucleic acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26. In some embodiments, the fibronectin polypeptide is encoded by a nucleic acid sequence of SEQ ID NO: 26. In some embodiments, the transferrin polypeptide or a functional fragment thereof comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 99% identical to SEQ ID NO: 27.
  • the transferrin polypeptide comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, the transferrin polypeptide comprises an amino acid sequence having at least one substitution (e.g., one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions and/or deletions) as compared to the corresponding wildtype transferrin polypeptide (e.g., wildtype human transferrin polypeptide, e.g., SEQ ID NO: 27).
  • the transferrin polypeptide can comprise one, two, three, four, five, six, seven, eight, nine, or ten of any type of substitution.
  • the transferrin polypeptide or a functional fragment thereof is encoded by a nucleic acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 28. In some embodiments, the transferrin polypeptide is encoded by a nucleic acid sequence of SEQ ID NO: 28.
  • the half-life extension moiety comprises an antibody or antigen- binding fragment thereof (e.g., any of the exemplary types of antigen-binding antibody fragments described herein or known in the art) that binds to a serum protein (e.g., albumin (e.g., human albumin), fibronectin (e.g., human fibronectin), or transferrin (e.g., human transferrin)).
  • a serum protein e.g., albumin (e.g., human albumin), fibronectin (e.g., human fibronectin), or transferrin (e.g., human transferrin)).
  • the half-life extension moiety comprises antigen-binding antibody fragments, e.g., single-chain antibodies (scFv), single-domain antibodies (also called nanobodies), Fab fragments, Fab ⁇ fragments, and F(ab ⁇ )2 Fab2 fragments, diabodies, triabodies, and tetrabodies, that bind to albumin (e.g., human albumin), fibronectin (e.g., human fibronectin), or transferrin (e.g., human transferrin).
  • the half-life extension moiety comprises a nanobody that binds specifically to human albumin, human fibronectin, or human transferrin.
  • Non-limiting examples of antibodies, antigen-binding fragments, and peptides that bind to a serum protein that can be used as half-life extension moieties are provided in WO 2013/128027; US 2015/0037334; US 9,376,495; US 8,349,569; US 10,407,508; and Michot, N., et al, Albumin binding Nanofitins, a new scaffold to extend half-life of biologics – a case study with exenatide peptide, Peptides, 152 (2022), the contents of each of which is incorporated herein by reference in its entirety.
  • the half-life extension moiety can comprise one or more post- translational modification site(s).
  • a post-translational modification site includes amino acid(s) in the polypeptide where a cellular enzyme can modify the amino acid to add a post-translational modification (e.g., phosphorylation, glycosylation, hydroxylation, methylation, acetylation, LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 deamidation, and lipidation (e.g., N-myristoylation, palmitoylation, glycosylphosphatidylinositol (GPI)-anchor addition) that results in an increase in the half-life of the polypeptide.
  • a post-translational modification e.g., phosphorylation, glycosylation, hydroxylation, methylation, acetylation, LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 deamidation
  • lipidation e.g., N-myristo
  • the polypeptide (encoded by the circular polyribonucleotides and modified RNAs described herein) can further comprise a secretion signal sequence.
  • secretion signal sequence refers to a polypeptide sequence (e.g., between about 10 and about 30 amino acids in length) that is present at the N-terminus of a protein and that targets at least a portion of the protein to the cellular secretory pathway.
  • a sequence encoding a secretion signal sequence (e.g., any of the exemplary secretion signal sequences described herein or known in the art) is operably linked to a sequence encoding a GLP-2 polypeptide or analog thereof (and a sequence encoding the half- life extension moiety).
  • the sequence encoding the secretion signal sequence can be operably linked at a 5’ end of a sequence encoding a GLP-2 polypeptide or analog thereof.
  • the secretion signal sequence is a liver-specific secretion signal sequence.
  • Non-limiting examples of a secretion signal sequence include an IL-2 secretion signal sequence, a proglucagon secretion signal sequence, a Gaussia lucifersase secretion signal sequence, and an erythropoietin (EPO) secretion signal sequence.
  • Non-limiting examples of amino acid sequences (and corresponding nucleic acid sequences) of an IL-2 secretion signal sequence, a proglucagon secretion signal sequence, a Gaussia lucifersase secretion signal sequence, and an EPO secretion signal sequence are shown in Table 4 below. Table 4.
  • Non-limiting examples of signal secretion sequences SEQ Description Sequence ID NO: C LRN Ref. No.: LRN23-107WO Fish Ref.
  • an IL-2 secretion signal sequence comprises an amino acid sequence of SEQ ID NO: 29.
  • a proglucagon secretion signal sequence comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 31.
  • a proglucagon secretion signal sequence comprises an amino acid sequence of SEQ ID NO: 31.
  • a Gaussia luciferase secretion signal sequence comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 33.
  • a Gaussia luciferase secretion signal sequence comprises an amino acid sequence of SEQ ID NO: 33.
  • an EPO secretion signal sequence comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 35.
  • an EPO secretion signal sequence comprises an amino acid sequence of SEQ ID NO: 35.
  • a signal secretion sequence comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, or 99% identical to one of SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO:
  • a signal secretion sequence comprises an amino acid sequence of SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91,
  • the sequence encoding a secretion signal sequence comprises a nucleic acid sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to one of SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36.
  • the sequence encoding a secretion signal sequence comprises a nucleic acid sequence of SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36.
  • the sequence encoding a secretion signal sequence comprises a nucleic acid sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to one of SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO:
  • the sequence encoding a secretion signal sequence comprises a nucleic acid sequence of SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 30
  • a “spacer” or “spacer sequence” or “spacer region” refers to any contiguous nucleotide sequence (e.g., of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions.
  • the circular polyribonucleotide or linear modified RNA as disclosed herein includes at least one spacer sequence.
  • the circular polyribonucleotide or linear modified RNA includes 1, 2, 3, 4, 5, 6, 7, or more spacer sequences (e.g., identical or different spacer sequences).
  • a first spacer sequence and a second spacer sequence are about the same length. In some embodiments, a first spacer sequence and a second spacer sequence (e.g., a third spacer sequence, a fourth spacer sequence, a fifth spacer sequence, etc.) are different lengths.
  • the spacer sequence can be a nucleic acid sequence having low GC content, e.g., less than 65%, 60%, 55%, 50%, 55%, 50%, 45%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31 %, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1 %, across the full length of the spacer, or across at least 50%, 60%, 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% contiguous nucleic acid residues of the spacer.
  • the spacer sequence is substantially free of a secondary structure, such as less than 40 kcal/mol, less than -39, -38, -37, -36, -35, -34, -33, -32, -31, -30, -29, -28, -27, -26, -25, -24, -23, -22, -20, - 19, -18, -17, -16, -15, -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2 or -1 kcal/mol.
  • a secondary structure such as less than 40 kcal/mol, less than -39, -38, -37, -36, -35, -34, -33, -32, -31, -30, -29, -28, -27, -26, -25, -24, -23, -22, -20, - 19, -18
  • the spacer sequence comprises: (i) a polyA region comprising 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) adenosine residues; (ii) a polyAC region comprising between 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) adenosine or cytosine residues; (iii) a polyAU region comprising between 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
  • the spacer sequence can be a non-coding sequence or a coding sequence.
  • a start codon may be provided in the coding sequence of an adjacent sequence.
  • a start codon may be provided in the spacer sequence.
  • the spacer is operably linked to another sequence described herein.
  • the circular polyribonucleotide or modified linear RNA includes an IRES operably linked to an expression sequence encoding a polypeptide.
  • the circular polyribonucleotide further includes a spacer region between the IRES and the 3’ exon fragment or the 5’ exon fragment.
  • the spacer region may be, e.g., at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length.
  • the spacer region may be, e.g., from 5 to 500 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500) ribonucleotides.
  • the spacer region includes a polyA sequence.
  • the spacer region includes a polyA-C sequence.
  • the spacer region includes a polyA-G sequence. In some embodiments, the spacer region includes a polyA- T sequence. In some embodiments, the spacer region includes a polyU sequence. In some embodiments, the spacer region includes a random sequence. In some embodiments, the spacer region includes a random sequence.
  • the circular polyribonucleotide is from 50 to 20,000, e.g., 100 to 20,000, e.g., 200 to 20,000, e.g., 300 to 20,000 (e.g., 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 3,500, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11 ,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000) ribonucleotides in length.
  • the circular polyribonucleotide is, e.g., at least 25, at least 50, at least 100, at least 125, at least 1150, at least 200, at least 300, at least 400, at least 500, at least 1,000, at least 2,000, at least 3,000, at least 4,000, or at least 5,000 ribonucleotides in length.
  • a spacer can be specifically engineered depending on the IRES.
  • an RNA folding computer software such as RNAFold, can be utilized to guide designs of the various elements of the vector, including the spacers.
  • the polyribonucleotide includes a 5’ spacer sequence (e.g., between the 5’ annealing region and the polyribonucleotide cargo).
  • the 5’ spacer sequence is at least 10 nucleotides in length.
  • the 5’ spacer LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 sequence is at least 15 nucleotides in length.
  • the 5’ spacer sequence is at least 30 nucleotides in length.
  • the 5’ spacer sequence is at least 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 nucleotides in length.
  • the 5’ spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some embodiments the 5’ spacer sequence is between 20 and 50 nucleotides in length. In certain embodiments, the 5’ spacer sequence is 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 3637, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In one embodiment, the 5’ spacer sequence is a polyA sequence. In another embodiment, the 5’ spacer sequence is a polyA-C sequence.
  • the 5’ spacer sequence includes a polyA-G sequence. In some embodiments, the 5’ spacer sequence includes a polyA-T sequence. In some embodiments, the 5’ spacer sequence includes a random sequence. In some embodiments, the polyribonucleotide includes a 3’ spacer sequence (e.g., between the 3’ annealing region and the polyribonucleotide cargo). In some embodiments, the 3’ spacer sequence is at least 10 nucleotides in length. In another embodiment, the 3’ spacer sequence is at least 15 nucleotides in length. In a further embodiment, the 3’ spacer sequence is at least 30 nucleotides in length.
  • the 3’ spacer sequence is at least 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 nucleotides in length. In some embodiments, the 3’ spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some embodiments the 3’ spacer sequence is from 20 to 50 nucleotides in length. In certain embodiments, the 3’ spacer sequence is 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length.
  • the 3’ spacer sequence is a polyA sequence.
  • the 5’ spacer sequence is a polyA-C sequence.
  • the 5’ spacer sequence includes a polyA-G sequence.
  • the 5’ spacer sequence includes a polyA-T sequence.
  • the 5’ spacer sequence includes a random sequence.
  • the polyribonucleotide includes a 5’ spacer sequence, but not a 3’ spacer sequence.
  • the polyribonucleotide includes a 3’ spacer sequence, but not a 5’ spacer sequence.
  • the polyribonucleotide includes neither a 5’ spacer sequence, nor a 3’ spacer sequence.
  • polyribonucleotide LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 does not include an IRES sequence.
  • polyribonucleotide does not include an IRES sequence, a 5’ spacer sequence or a 3’ spacer sequence.
  • the spacer sequence includes at least 3 ribonucleotides, at least 4 ribonucleotides, at least 5 ribonucleotides, at least about 8 ribonucleotides, at least about 10 ribonucleotides, at least about 12 ribonucleotides, at least about 15 ribonucleotides, at least about 20 ribonucleotides, at least about 25 ribonucleotides, at least about 30 ribonucleotides, at least about 40 ribonucleotides, at least about 50 ribonucleotides, at least about 60 ribonucleotides, at least about 70 ribonucleotides, at least about 80 ribonucleotides, at least about 90 ribonucleotides, at least about 100 ribonucleotides, at least about 120 ribonucleotides, at least about 150 ribonucleotides, at least 3 rib
  • the circular polyribonucleotide or modified RNA further comprises a sequence encoding a linker sequence.
  • the circular polyribonucleotide or modified RNA can comprise a sequence encoding a linker sequence disposed between the sequence encoding the GLP-2 polypeptide or analog thereof and the sequence encoding the half- life extension moiety.
  • the polypeptide comprises a linker sequence disposed between the GLP-2 polypeptide or analog thereof and the half-life extension moiety in the polypeptide.
  • linker sequences are shown in Table 6 below. Additional examples of linker sequences are well known in the art (See e.g., Chen, X., Fusion Protein Linkers: Property, Design and Functionality, Adv Drug Deliv Rev., 65(10): 1357-1369 (2013), the content of which is incorporated herein by reference in its entirety). Table 6.
  • Non-limiting examples of linker sequences SEQ ID Description Sequence NO: LRN Ref. No.: LRN23-107WO Fish Ref.
  • any of the circular polyribonucleotides or elements thereof described herein can include one or more modifications (e.g., any of the exemplary modifications described herein or known in the art).
  • Any of the modified RNAs described herein can include one or more modifications (e.g., any of the exemplary modifications described herein or known in the art).
  • the one or more modifications can be included at any position in the circular polyribonucleotide or modified RNA.
  • the sequence encoding a GLP-2 polypeptide or analog thereof can comprise one or more modifications (e.g., the sequence encoding a GLP-2 polypeptide or analog thereof comprises one or more modified bases).
  • the circular polyribonucleotide or modified RNA can comprise a post-transcriptional modification (e.g., capping, cleavage, polyadenylation, and splicing).
  • a circular polyribonucleotide or elements thereof, or a modified RNA or elements thereof can include one or more nucleoside modifications (e.g., any of those described in Rozenski et al., Nucl. Acids Res. 27: 196-197, 1999).
  • the circular polyribonucleotide or modified RNA comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) modification(s) to a sugar, a nucleobase, an internucleoside linkage, or a combination thereof.
  • the circular polyribonucleotide or modified RNA can include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) pyrimidine nucleobases having one or more atoms substituted with an amino, a thiol, an alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro) group.
  • pyrimidine nucleobases having one or more atoms substituted with an amino, a thiol, an alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro) group.
  • the circular polyribonucleotide or modified RNA can include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ribonucleotides that have been modified into deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), and/or locked nucleic acids (LNAs).
  • the circular polyribonucleotide or modified RNA includes at least one (e.g., two, three, four, or five) N(6)methyladenosine (m6A) modification to increase translation efficiency.
  • m6A N(6)methyladenosine
  • the N(6)methyladenosine (m6A) modification can LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 reduce immunogenicity (e.g., reduce the level of one or more marker of an immune or inflammatory response) of the circular polyribonucleotide or modified RNA.
  • a modification may include a chemical or cellular induced modification.
  • intracellular RNA modifications are described in Lewis et al., Nature Reviews Mol. Cell Biol. 18:202-210, 2017, herein incorporated by reference.
  • chemical modifications to the ribonucleotides of a circular polyribonucleotide or modified RNA may enhance immune evasion.
  • the circular polyribonucleotide or modified RNA may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, herein incorporated by reference.
  • Modifications include, for example, end modifications, e.g., 5’ end modifications (phosphorylation (mono-, di- and tri-), conjugation, inverted linkages), 3’ end modifications (conjugation, DNA nucleotides, inverted linkages), base modifications (e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners), removal of bases (abasic nucleotides), or conjugated bases.
  • the modified ribonucleotide bases may also include 5- methylcytidine, N1-methylpseudouridine, 5- methoxyuridine, and pseudouridine.
  • base modifications may modulate expression, immune response, stability, subcellular localization, to name a few functional effects, of the circular polyribonucleotide or modified RNA.
  • the modification includes a bi-orthogonal nucleotide, e.g., an unnatural base. See, e.g., Kimoto et al., Chem. Comm. (Camb) 53:12309, 2017, which is herein incorporated by reference.
  • one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ribonucleotides of the circular polyribonucleotide or modified RNA can include a sugar modification (e.g., at the 2’ position or 4’ position) and optionally, a backbone modification, including modification or replacement of the phosphodiester linkages.
  • a sugar modification e.g., at the 2’ position or 4’ position
  • a backbone modification including modification or replacement of the phosphodiester linkages.
  • Specific examples of circular polyribonucleotides or modified RNAs include, but are not limited to, circular polyribonucleotides or modified RNAs including modified backbones or no natural internucleoside linkages, such as internucleoside modifications, including modification or replacement of the phosphodiester linkages.
  • Circular polyribonucleotides and modified RNAs having modified backbones include, among others, those that do not have a phosphorus atom in LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 the backbone.
  • the circular polyribonucleotide or modified RNA will include ribonucleotides with a phosphorus atom in its internucleoside backbone.
  • Modified backbones of circular polyribonucleotides and modified RNAs can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, such as 3’-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, such as 3’-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'- 5’ linkages, 2’-5’ linked analogs of these, and those having inverted polarity, wherein the adjacent pairs of nucleoside units are linked 3’-5’ to 5’-3’ or 2’-5’ to 5’-2’.
  • the circular polyribonucleotides or modified RNAs may be negatively- or positively-charged.
  • the modified nucleotide(s), which may be incorporated into the circular polyribonucleotides or modified RNAs, can be modified on the internucleoside linkage (e.g., phosphate backbone).
  • phosphate backbone the phrases “phosphate” and “phosphodiester” are used interchangeably.
  • Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent.
  • modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein.
  • modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters.
  • Phosphorodithioates have both non-linking oxygens replaced by sulfur.
  • the phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene -phosphonates).
  • A-thio substituted phosphate moieties can be included to confer stability to circular polyribonucleotides and modified RNAs through the unnatural phosphorothioate backbone linkages.
  • Phosphorothioate RNAs have increased nuclease resistance and subsequently a longer half-life in a cellular environment.
  • a modified nucleoside can include an alpha-thio-nucleoside (e.g., 5’-O-(l-thiophosphate)-adenosine, 5’-O-(l-thiophosphate)-cytidine (a-thio-cytidine), 5’-O- (l-thiophosphate)-guanosine, 5’-O-(l-thiophosphate)-uridine, or 5’-O- (1-thiophosphate)- pseudouridine).
  • alpha-thio-nucleoside e.g., 5’-O-(l-thiophosphate)-adenosine, 5’-O-(l-thiophosphate)-cytidine (a-thio-cytidine), 5’-O- (l-thiophosphate)-guanosine, 5’-O-(l-thiophosphate)-uridine, or 5’-O- (1-thiophosphate)- pseudouridine).
  • a circular polyribonucleotide or modified RNA may or may not be uniformly modified along the entire length of the molecule.
  • nucleotide e.g., naturally-occurring nucleotides, purine or pyrimidine, or any one or more or all of A, G, U, C, I, pU
  • the circular polyribonucleotide or modified RNA includes a pseudouridine.
  • the circular polyribonucleotide or modified RNA includes an inosine, which may aid in the immune system characterizing the circular polyribonucleotide or modified RNA as an endogenous RNA versus a viral RNA.
  • the incorporation of inosine may also mediate improved RNA stability/reduced degradation. See for example, Yu, Z. et al., Cell Res. 25, 1283–1284, 2015, which is incorporated by reference in its entirety.
  • all or substantially all nucleotides in a circular polyribonucleotide or modified RNA are modified.
  • the modification may include an m6A, which may augment expression; an inosine, which may attenuate an immune response; pseudouridine, which may increase RNA stability, or translational readthrough (stagger element); an m5C, which may increase stability; and a 2,2,7- trimethylguanosine, which aids subcellular translocation (e.g., nuclear localization).
  • m6A which may augment expression
  • pseudouridine which may increase RNA stability, or translational readthrough (stagger element)
  • an m5C which may increase stability
  • a 2,2,7- trimethylguanosine which aids subcellular translocation (e.g., nuclear localization).
  • Different sugar modifications, nucleotide modifications, and/or internucleoside linkages may exist at various positions in a circular polyribonucleotide or modified RNA.
  • nucleotide analogs or other modification(s) may be located at any position(s) of the circular polyribonucleotide or modified RNA, such that the function of the circular polyribonucleotide or modified RNA is not LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 substantially decreased.
  • a modification can be included in a coding sequence or a non-coding sequence.
  • the circular polyribonucleotide or modified RNA may include about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, e.g., any one or more of A, G, U, or C) or any intervening percentage (e.g., about 1% to about 10%, about 1% to about 15%, about 1% to about 20%, from about 1% to about 25%, about 1% to about 30%, about 1% to about 40%, about 1% to about 50%, about 1% to about 60%, about 1% to about 70%, about 1% to about 80%, about 1% to about 90%, about 1% to about 95%, about 10% to about 20%, about 10% to about 25%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 95%, about 10% to about 100%, about 20% to about 25%, about 20% to about 25%
  • the circular polyribonucleotides or modified RNAs described herein includes one or more internal ribosome entry site(s) (IRES(s)).
  • an IRES is operably linked to the sequence encoding the polypeptide (e.g., any of the exemplary polypeptides described herein).
  • an IRES is located at or proximal to the LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 5’ end of the sequence encoding the polypeptide (e.g., any of the exemplary polypeptides described herein).
  • a suitable IRES includes an RNA sequence capable of engaging a eukaryotic ribosome.
  • an IRES is at least about 50 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 250 nucleotides, at least about 350 nucleotides, or at least about 500 nucleotides.
  • an IRES is about 50 nucleotides to about 500 nucleotides, about 50 nucleotides to about 450 nucleotides, about 50 nucleotides to about 400 nucleotides, about 50 nucleotides to about 350 nucleotides, about 50 nucleotides to about 300 nucleotides, about 50 nucleotides to about 250 nucleotides, about 50 nucleotides to about 200 nucleotides, about 50 nucleotides to about 150 nucleotides, about 50 nucleotides to about 100 nucleotides, about 100 nucleotides to about 500 nucleotides, about 100 nucleotides to about 450 nucleotides, about 100 nucleotides to about 400 nucleotides, about 100 nucleotides to about 350 nucleotides, about 100 nucleotides to about 300 nucleotides, about 100 nucleotides to about 250 nucleotides, about 100 nucle
  • the IRES is derived from the DNA of an organism including, but not limited to, a virus, a mammal, and Drosophila.
  • a virus a mammal
  • Drosophila Such viral DNA may be derived from, but LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 not limited to, picornavirus complementary DNA (cDNA), encephalomyocarditis virus (EMCV) cDNA, and poliovirus cDNA.
  • Drosophila DNA from which an IRES is derived includes, but is not limited to, an Antennapedia gene from Drosophila melanogaster.
  • nucleic acid in the context of a nucleic acid, i.e., for a nucleic acid “derived from” (another) nucleic acid, means that the nucleic acid, which is derived from (another) nucleic acid, shares e.g. at least 60%, 70%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid from which it is derived.
  • sequence identity is typically calculated for the same types of nucleic acids, i.e., for DNA sequences or for RNA sequences.
  • a DNA is “derived from” an RNA or if an RNA is “derived from” a DNA
  • the RNA sequence is converted into the corresponding DNA sequence (in particular by replacing the uracils (U) by thymidines (T) throughout the sequence) or, vice versa
  • the DNA sequence is converted into the corresponding RNA sequence (in particular by replacing the T by U throughout the sequence).
  • sequence identity of the DNA sequences or the sequence identity of the RNA sequences is determined.
  • nucleic acid “derived from” a nucleic acid also refers to nucleic acid, which is modified in comparison to the nucleic acid from which it is derived, e.g., in order to increase RNA stability even further and/or to prolong and/or increase protein production.
  • the term “derived from” means that the amino acid sequence, which is derived from (another) amino acid sequence, shares e.g.
  • the IRES is an IRES of Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, human poliovirus 1, Plautia stall intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus- 1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus- 1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus (EMCV), Drosophila C Virus, Crucifer tobamo virus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus,
  • EMCV Encephal
  • the IRES is an IRES of Coxsackievirus B3 (CVB3).
  • the IRES is an IRES of Encephalomyocarditis virus.
  • the IRES sequence has more than 90% sequence identify with one of the foregoing IRES sequences.
  • the IRES sequence may have the sequence of wild-type CVB3 IRES sequence having the nucleic acid sequence of: TTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGG TATCACGGTACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAG TAACACACACCGATCAACAGTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCAC TTCTGTTACCCCGGACTGAGTATCAATAGACTGCTCACGCGGTTGAAGGAGAAAGCG TTCGTTATCCGGCCAACTACTTCGAAAAACCTAGTAACACCGTGGAAGTTGCAGAGT GTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCATTCCCCAC GGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGAAACCCATGGGAC GCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCGGCCGGCCGGGG
  • the IRES sequence may have a modified sequence in comparison to the wildtype IRES sequence.
  • the last nucleotide of the wild-type IRES when the last nucleotide of the wild-type IRES is not a cytosine nucleic acid residue, the last nucleotide of the wild-type IRES sequence may be modified such that it is a cytosine residue.
  • the IRES sequence may be a CVB3 IRES sequence wherein the terminal adenosine residue is modified to cytosine residue.
  • the modified CVB3 IRES may have the nucleic acid sequence of: TTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGG TATCACGGTACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAG TAACACACACCGATCAACAGTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCAC TTCTGTTACCCCGGACTGAGTATCAATAGACTGCTCACGCGGTTGAAGGAGAAAGCG TTCGTTATCCGGCCAACTACTTCGAAAAACCTAGTAACACCGTGGAAGTTGCAGAGT GTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCATTCCCCAC GGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGAAACCCATGGGGAAACCCATGGGAC GCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCGGCC CC CCTGAATGCGGC
  • the modified CVB3 IRES may have the nucleic acid sequence of: CCAACTGTAACTTAGAAGTAACACACACCGATCAACAGTCAGCGTGGCACACCAGC CACGTTTTGATCAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGACTGCTCACG CGGTTGAAGGAGAAAGCGTTCGTTATCCGGCCAACTACTTCGAAAAACCTAGTAAC ACCGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGAT GAGTCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCC ATGGGGAAACCCATGGGACGCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGC TAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACACACC CTCAAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACT TTGGGTGTCCGTGTTTCATTTTATTATTGGGTAG
  • the IRES sequence is an encephalomyocarditis virus (EMCV) IRES.
  • the ECMV IRES may have the nucleic acid sequence of: ACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTAT TTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTT CTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTT GAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGT AGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCC AAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTT GTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAG GGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCG GTGCACATGCTTTACATGTGTTTAGTC
  • the IRES sequence is an Enterovirus 71 (EV71) IRES.
  • the IRES sequence is a wild-type EV71 sequence having the nucleic acid sequence of: TTAAAACAGCTGTGGGTTGTCACCCACCCACAGGGTCCACTGGGCGCTAGTACACTG GTATCTCGGTACCTTTGTACGCCTGTTTTATACCCCCTCCCTGATTTGCAACTTAGAA GCAACGCAAACCAGATCAATAGTAGGTGTGACATACCAGTCGCATCTTGATCAAGC ACTTCTGTATCCCCGGACCGAGTATCAATAGACTGTGCACACGGTTGAAGGAAA ACGTCCGTTACCCGGCTAACTACTTCGAGAAGCCTAGTAACGCCATTGAAGTTGCAG AGTGTTTCGCTCAGCACTCCCCCCGTGTAGATCAGGTCGATGAGTCACCGCATTCCC CACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCTATGGGGTAACCCATAG GACGCTCTAATACGGACATGGCGACCGTGGCTGCGTTGCC
  • the terminal guanosine residue of the EV71 IRES sequence is modified to a cytosine residue.
  • the polyribonucleotide includes at least one IRES flanking at least one (e.g., 2, 3, 4, 5 or more) expression sequence. In some embodiments, the IRES flanks both sides of at least one (e.g., 2, 3, 4, 5 or more) expression sequence. In some embodiments, the polyribonucleotide includes one or more IRES sequences on one or both sides of each expression sequence, leading to separation of the resulting peptide(s) and or polypeptide(s).
  • a polyribonucleotide described herein may include a first IRES operably linked to a first expression sequence and a second IRES operably linked to a second expression sequence.
  • an exemplary construct described above includes a construct with two IRES sequences each of which is followed by an ORF sequence.
  • a spacer sequence is disposed between the first IRES-ORF sequence and the second IRES-ORF sequence.
  • both the first ORF and the second ORF sequence encode a GLP-2 polypeptide or analog thereof (e.g., GLP-2 Fc).
  • the first ORF encodes a GLP-2 polypeptide or analog thereof (e.g., GLP-2 Fc) and the second ORF encodes a different polypeptide.
  • the circular polyribonucleotides or modified RNAs described herein can include a modified IRES, such as those described in WO 2020/198403, which is incorporated herein by reference in its entirety or an IRES, such as those described in Fan et al. Nature Communications 13(1):3751, 2022 doi: 10.1038/s41467-022-31327-y; Chen et al. Mol. Cell 81:1-19, 2021; Jopling et al.
  • the circular polyribonucleotides or modified RNAs described herein can include at least one translation initiation sequence.
  • the circular polyribonucleotide or modified RNA includes a translation initiation sequence operably linked to LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 the sequence encoding the polypeptide (e.g., any of the exemplary polypeptides described herein).
  • the translation initiation sequence includes, e.g., a start codon.
  • the translation initiation sequence includes a Kozak or Shine-Dalgarno sequence.
  • the circular polyribonucleotide or modified RNA includes the translation initiation sequence, e.g., Kozak sequence, adjacent to the sequence encoding the polypeptide (e.g., any of the exemplary polypeptides described herein).
  • the translation initiation sequence e.g., Kozak sequence
  • the circular polyribonucleotide or modified RNA includes at least one translation initiation sequence adjacent to the sequence encoding the polypeptide (e.g., any of the exemplary polypeptides described herein).
  • the translation initiation sequence provides conformational flexibility to the circular polyribonucleotide or modified RNA. In some embodiments, the translation initiation sequence is within a substantially single stranded region of the circular polyribonucleotide or modified RNA.
  • the circular polyribonucleotide may include more than 1 start codon such as, but not limited to, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, or more than 60 start codons.
  • translation may initiate on the first start codon or may initiate downstream of the first start codon.
  • the circular polyribonucleotide or modified RNA may initiate at a codon that is not the first start codon, e.g., AUG.
  • Translation of the circular polyribonucleotide or modified RNA may initiate at an alternative translation initiation sequence, such as, but not limited to, ACG, AGG, AAG, CTG/CUG, GTG/GUG, ATA/AUA, ATT/AUU, or TTG/UUG.
  • translation begins at an alternative translation initiation sequence under selective conditions, e.g., stress-induced conditions.
  • the translation of the circular polyribonucleotide or modified RNA may begin at alternative translation initiation sequence, such as ACG.
  • alternative translation initiation sequence such as ACG.
  • the circular polyribonucleotide or modified RNA translation may begin at alternative translation initiation sequence, CTG/CUG.
  • the circular polyribonucleotide or modified RNA translation may begin at alternative translation initiation sequence, GTG/GUG.
  • the circular polyribonucleotide or modified RNA may begin translation at a repeat-associated non-AUG (RAN) sequence, such as an alternative translation initiation sequence that includes short stretches of repetitive RNA, e.g., CGG, GGGGCC, CAG, or CTG.
  • Nucleotides flanking a codon that initiates translation may affect the translation efficiency and the length and/or the structure of the circular polyribonucleotide or modified RNA. Masking any of the nucleotides flanking a codon that initiates translation may be used to alter the position of translation initiation, translation efficiency, and length, and/or structure of the circular polyribonucleotide or modified RNA.
  • a masking agent may be used near the start codon or alternative start codon in order to mask or hide the codon to reduce the probability of translation initiation at the masked start codon or alternative start codon.
  • masking agents include antisense locked nucleic acids (LNA) oligonucleotides and exon-junction complexes (EJCs).
  • LNA antisense locked nucleic acids
  • EJCs exon-junction complexes
  • a masking agent may be used to mask a start codon of the circular polyribonucleotide or modified RNA in order to increase the likelihood that translation will initiate at an alternative start codon.
  • translation is initiated under selective conditions, such as, but not limited to, viral-induced selection in the presence of GRSF-1 and the circular polyribonucleotide or modified RNA includes GRSF-1 binding sites.
  • translation is initiated by eukaryotic initiation factor 4A (eIF4A) treatment with Rocaglates. Translation may be repressed by blocking 43S scanning, leading to premature, upstream translation initiation and reduced protein expression from transcripts bearing the RocA–eIF4A target sequence.
  • eIF4A eukaryotic initiation factor 4A
  • translation may be repressed by blocking 43S scanning, leading to premature, upstream translation initiation and reduced protein expression from transcripts bearing the RocA–eIF4A target sequence.
  • the circular polyribonucleotides or modified RNAs can further include at least one translation termination sequence.
  • the translation termination sequence is operably linked to the 3’ end of the sequence encoding the polypeptide (e.g., any of the exemplary polypeptides described herein).
  • the circular polyribonucleotide or modified RNA includes a termination element at the end of a sequence encoding the polypeptide (e.g., any of the polypeptides described herein).
  • a circular polyribonucleotide or modified RNA can include two or more termination elements in succession.
  • translation termination sequences include an in-frame nucleotide triplet that signals termination of translation, e.g., UAA, UGA, or UAG.
  • one or more translation termination sequences in the circular polyribonucleotide or modified RNA are frame-shifted termination elements, such as but not limited to, off-frame or -1 and + 1 shifted reading frames (e.g., hidden stop) that may terminate translation.
  • Frame-shifted termination elements include nucleotide triples, e.g., TAA, TAG, or TGA, that appear in the second and third reading frames of the sequence encoding the polypeptide.
  • Frame-shifted termination elements may be important in preventing misreads of the sequence encoding the polypeptide, which is often detrimental to a cell.
  • the termination element is a stop codon.
  • Further examples of translation termination sequences are described in paragraphs [0169] - [0170] of WO2019/118919, which is hereby incorporated by reference in its entirety.
  • Stagger Elements A circular polyribonucleotide of the disclosure can include a cleavage domain (e.g., a stagger element or a cleavage sequence).
  • the circular polyribonucleotide includes a stagger sequence or a stagger element.
  • stagger element or a “stagger sequence” is a moiety, such as a nucleotide sequence, that induces ribosomal pausing during translation.
  • a stagger sequence can be included to induce ribosomal pausing during translation.
  • the stagger sequence may include a 2A-like or CHYSEL (cis-acting hydrolase element) sequence.
  • the stagger element encodes a sequence with a C- terminal consensus sequence that is X 1 X 2 X 3 EX 5 NPGP (SEQ ID NO: 144), where X 1 is absent or G or H, X2 is absent or D or G, X3 is D, V, I, S, or M, and X5 is any amino acid.
  • the stagger element comprises a non-conserved sequence of amino-acids with a strong alpha-helical propensity followed by the consensus sequence -D(V/I)EXNPGP (SEQ ID LRN Ref. No.: LRN23-107WO Fish Ref.
  • stagger elements include any of the stagger sequences shown in Table 7.
  • a stagger element described herein terminates translation and/or cleaves an expression product between G and P of the consensus sequence described herein.
  • the circular polyribonucleotide or modified RNA includes at least one stagger sequence to terminate translation and/or cleave the encoded polypeptide.
  • the circular polyribonucleotide or modified RNA includes a stagger element adjacent to at least one sequence encoding a polypeptide (e.g., any of the exemplary polypeptides described herein).
  • the circular polyribonucleotide or modified RNA includes a stagger element after each sequence encoding a polypeptide (e.g., any of the exemplary polypeptides described herein). In some embodiments, the circular polyribonucleotide or modified RNA includes a stagger element present on one or both sides of each sequence encoding a polypeptide (e.g., any of the exemplary polypeptides described herein), leading to translation of individual peptide(s) and or polypeptide(s) from each expression sequence.
  • Table 7 Non-limiting examples of stagger elements SEQ Description Sequence ID LRN Ref. No.: LRN23-107WO Fish Ref.
  • the stagger element may include a chemical moiety, such as glycerol, a non-nucleic acid linking moiety, a chemical modification, a modified nucleic acid, or any combination thereof.
  • Replication Elements Replication of a circular polyribonucleotide can occur by generating a complement circular polyribonucleotide.
  • the circular polyribonucleotide can include a motif to initiate transcription, where transcription is driven by either endogenous cellular machinery (DNA-dependent RNA polymerase) or an RNA-depended RNA polymerase encoded by the circular polyribonucleotide.
  • the product of the rolling-circle transcriptional event can be cut by a ribozyme to generate either complementary or propagated circular polyribonucleotide at unit length.
  • the ribozyme can be encoded by the circular polyribonucleotide, its complement, or by an RNA sequence in trans.
  • the encoded ribozyme can include a sequence or motif that regulates (inhibits or promotes) activity of the ribozyme to control circular polyribonucleotide propagation.
  • unit-length sequences can be ligated into a circular form by a cellular RNA ligase.
  • the circular polyribonucleotide includes a replication element that aids in self-amplification.
  • replication elements include HDV replication domains and replication competent circular RNA sense and/or antisense ribozymes, such as antigenomic 5’- CGGGUCGGCAUGGCAUCUCCACCUCCUCGC GGUCCGACCUGGGCAUCCGAAGGAGGACGCACGUCCACUCGGAUGGCUAAGGGAG LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 AGCCA-3’ (SEQ ID NO: 165) or genomic 5’- UGGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUU CCGAGGGGACCGUCCCCUCGGUAAUGGCGAAUGGGACCCA-3’ (SEQ ID NO: 166).
  • antigenomic 5 - CGGGUCGGCAUGGCAUCUCCACCUCCUCGC GGUCCGACCUGGGCAUCCGAAGGAGGACGCACGUCCACUCGGAUGGCUAAGGGAG LRN Ref. No.: LRN23-107WO Fish Ref. No
  • the circular polyribonucleotide includes at least one cleavage sequence as described herein to aid in replication.
  • a cleavage sequence within the circular polyribonucleotide can cleave long transcripts replicated from the circular polyribonucleotide to a specific length that can subsequently circularize to form a complement to the circular polyribonucleotide.
  • the circular polyribonucleotide includes at least one ribozyme sequence to cleave long transcripts replicated from the circular polyribonucleotide to a specific length, where another encoded ribozyme cuts the transcripts at the ribozyme sequence. Circularization forms a complement to the circular polyribonucleotide.
  • the circular polyribonucleotide is substantially resistant to degradation, e.g., by exonucleases.
  • a “substantially resistant” circular polyribonucleotide can refer to one that has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% resistance as compared to a reference polyribonucleotide.
  • the circular polyribonucleotide replicates within a cell.
  • the circular polyribonucleotide replicates within in a cell at a rate of about 10%- 20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-99%, or any percentage therebetween.
  • the circular polyribonucleotide replicates within a cell and is passed to daughter cells.
  • a cell passes at least one circular polyribonucleotide to daughter cells with an efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%.
  • a cell undergoing meiosis passes the circular polyribonucleotide to daughter cells with an efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, a cell undergoing mitosis passes the circular polyribonucleotide to daughter cells with an efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the circular polyribonucleotide replicates within the host cell. In some embodiments, the circular polyribonucleotide is capable of replicating in a mammalian cell, e.g., a human cell. LRN Ref.
  • the circular polyribonucleotide replicates in the host cell, the circular polyribonucleotide does not integrate into the genome of the host, e.g., with the host’s chromosomes. In some embodiments, the circular polyribonucleotide has a negligible recombination frequency, e.g., with the host’s chromosomes.
  • the circular polyribonucleotide has a recombination frequency, e.g., less than about 1.0 cM/Mb, 0.9 cM/Mb, 0.8 cM/Mb, 0.7 cM/Mb, 0.6 cM/Mb, 0.5 cM/Mb, 0.4 cM/Mb, 0.3 cM/Mb, 0.2 cM/Mb, 0.1 cM/Mb, or less, e.g., with the host’s chromosomes.
  • a recombination frequency e.g., less than about 1.0 cM/Mb, 0.9 cM/Mb, 0.8 cM/Mb, 0.7 cM/Mb, 0.6 cM/Mb, 0.5 cM/Mb, 0.4 cM/Mb, 0.3 cM/Mb, 0.2 cM/Mb, 0.1 cM/Mb, or less, e.
  • a DNA molecule used to produce a circular RNA molecule can include a DNA sequence of a naturally occurring nucleic acid sequence, a modified version thereof, or a DNA sequence encoding a synthetic polypeptide not normally found in nature (e.g., chimeric molecules or fusion proteins).
  • DNA and RNA molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant techniques, such as site- directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof.
  • the circular polyribonucleotides may be prepared according to any available technique, including, but not limited to chemical synthesis and enzymatic synthesis.
  • a linear primary construct or linear polyribonucleotide for circularization may be cyclized or concatenated to create a circular RNA described herein.
  • the linear polyribonucleotide for circularization may be cyclized in vitro prior to formulation and/or delivery.
  • the circular polyribonucleotide may be in a mixture with linear polyribonucleotides.
  • the linear polyribonucleotides have the same nucleic acid sequence as the circular polyribonucleotides.
  • the mechanism of cyclization or concatenation may occur through methods such as, e.g., chemical, enzymatic, splint ligation, or ribozyme-catalyzed methods.
  • the newly formed 5’-3’ LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 linkage may be an intramolecular linkage or an intermolecular linkage.
  • a splint ligase such as a SplintR® ligase, can be used for splint ligation.
  • a single stranded polynucleotide such as a single-stranded DNA or RNA
  • splint can be designed to hybridize with both termini of a linear polyribonucleotide, so that the two termini can be juxtaposed upon hybridization with the single-stranded splint.
  • Splint ligase can thus catalyze the ligation of the juxtaposed two termini of the linear polyribonucleotide, generating a circular polyribonucleotide.
  • a DNA or RNA ligase may be used in the synthesis of the circular polynucleotides.
  • the ligase may be a circ ligase or circular ligase.
  • either the 5' or 3' end of the linear polyribonucleotide can encode a ligase ribozyme sequence such that during in vitro transcription, the resultant linear polyribonucleotide for circularization includes an active ribozyme sequence capable of ligating the 5' end of the linear polyribonucleotide for circularization to the 3' end of the linear polyribonucleotide for circularization.
  • the ligase ribozyme may be derived from the Group I Intron, Hepatitis Delta Virus, Hairpin ribozyme or may be selected by SELEX (systematic evolution of ligands by exponential enrichment).
  • a linear polyribonucleotide may be cyclized or concatenated by using at least one non-nucleic acid moiety.
  • the at least one non-nucleic acid moiety may react with regions or features near the 5' terminus or near the 3' terminus of the linear polyribonucleotide for circularization in order to cyclize or concatenate the linear polyribonucleotide for circularization.
  • the at least one non-nucleic acid moiety may be located in or linked to or near the 5' terminus or the 3' terminus of the linear polyribonucleotide for circularization.
  • the non-nucleic acid moieties may be homologous or heterologous.
  • the non-nucleic acid moiety may be a linkage such as a hydrophobic linkage, ionic linkage, a biodegradable linkage, or a cleavable linkage.
  • the non-nucleic acid moiety is a ligation moiety.
  • the non-nucleic acid moiety may be an oligonucleotide or a peptide moiety, such as an aptamer or a non-nucleic acid linker as described herein.
  • a linear polyribonucleotide for circularization may include a 5' triphosphate of the nucleic acid converted into a 5' monophosphate, e.g., by contacting the 5' triphosphate with RNA 5' pyrophosphohydrolase (RppH) or an ATP diphosphohydrolase LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 (apyrase).
  • the 5’ end of at least a portion of the linear polyribonucleotides includes a monophosphate moiety.
  • a population of polyribonucleotides including circular and linear polyribonucleotides is contacted with RppH prior to digesting at least a portion of the linear polyribonucleotides with a 5’ exonuclease and/or a 3’ exonuclease.
  • converting the 5' triphosphate of the linear polyribonucleotide for circularization into a 5' monophosphate may occur by a two-step reaction including: (a) contacting the 5' nucleotide of the linear polyribonucleotide for circularization with a phosphatase (e.g., Antarctic Phosphatase, Shrimp Alkaline Phosphatase, or Calf Intestinal Phosphatase) to remove all three phosphates; and (b) contacting the 5' nucleotide after step (a) with a kinase (e.g., Polynucleotide Kinase) that adds a single phosphate.
  • a phosphatase e.g., Antarctic Phosphatase, Shrimp Alkaline Phosphatase, or Calf Intestinal Phosphatase
  • a kinase e.g., Polynucleotide
  • linear polyribonucleotides for circularization may be cyclized or concatenated by self-splicing.
  • the linear polyribonucleotide may include a sequence that mediates self- ligation.
  • the linear polyribonucleotides may include loop E sequence (e.g., in PSTVd) to self-ligate.
  • the linear polyribonucleotide may include a HDV sequence, e.g., HDV replication domain conserved sequence, GGCUCAUCUCGACAAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUCUGUAAAG AGGAGACUGCUGGACUCGCCGCCCAAGUUCGAGCAUGAGCC (SEQ ID NO: 170) or GGCUAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUCUGUAAAGAGGAGACUG CUGGACUCGCCGCCCGAGCC (SEQ ID NO: 171), to self-ligate.
  • a HDV sequence e.g., HDV replication domain conserved sequence, GGCUCAUCUCGACAAGAGGCGGCAGUCCUCAGUACUCUUACUUUUCUGUAAAG AGGAGACUGCUGGACUCGCCGCCCAAGUUCGAGCAUGAGCC (SEQ ID NO: 170) or GGCUAGAGGCGGCAGUCCUCAGUACUCUUACUUUUCUGUAAAGAGGAGAC
  • the linear polyribonucleotides may include a self-circularizing intron, e.g., a 5' and 3’ slice junction, or a self-circularizing catalytic intron such as a Group I, Group II, or Group III Introns.
  • group I intron self- splicing sequences may include self-splicing permuted intron-exon sequences derived from T4 bacteriophage gene td, and the intervening sequence (IVS) rRNA of Tetrahymena, cyanobacterium Anabaena pre-tRNA-Leu gene, or a Tetrahymena pre-rRNA.
  • the polyribonucleotide includes catalytic intron fragments, such as a 3′ half of Group I catalytic intron fragment and a 5′ half of Group I catalytic intron fragment.
  • the first and second annealing regions may be positioned within the catalytic intron fragments.
  • Group I catalytic introns are self-splicing ribozymes that catalyze their own excision from mRNA, tRNA, and rRNA precursors via two-metal ion phorphoryl transfer mechanism. LRN Ref. No.: LRN23-107WO Fish Ref.
  • the RNA itself self-catalyzes the intron removal without the requirement of an exogenous enzyme, such as a ligase.
  • the 3′ half of Group I catalytic intron fragment and the 5’ half of Group I catalytic intron fragment are from a cyanobacterium Anabaena pre-tRNA-Leu gene, or a Tetrahymena pre-rRNA.
  • the 3′ half of Group I catalytic intron fragment and the 5’ half of Group I catalytic intron fragment are from a Cyanobacterium Anabaena pre-tRNA-Leu gene, and the 3’ exon fragment includes the first annealing region and the 5’ exon fragment includes the second annealing region.
  • the first annealing region may include, e.g., from 5 to 50, e.g., from 10 to 15 (e.g., 10, 11, 12, 13, 14, or 15) ribonucleotides and the second annealing region may include, e.g., from 5 to 50, e.g., from 10 to 15 (e.g., 10, 11, 12, 13, 14, or 15) ribonucleotides.
  • the 3′ half of Group I catalytic intron fragment and the 5’ half of Group I catalytic intron fragment are from a Tetrahymena pre-rRNA, and the 3′ half of Group I catalytic intron fragment includes the first annealing region and the 5’ exon fragment includes the second annealing region. In some embodiments, the 3′ exon includes the first annealing region and the 5’ half of Group I catalytic intron fragment includes the second annealing region.
  • the first annealing region may include, e.g., from 6 to 50, e.g., from 10 to 16 (e.g., 10, 11, 12, 13, 14, 15, or 16) ribonucleotides
  • the second annealing region may include, e.g., from 6 to 50, e.g., from 10 to 16 (e.g., 10, 11, 12, 13, 14, 15, or 16) ribonucleotides.
  • the 3′ half of Group I catalytic intron fragment and the 5’ half of Group I catalytic intron fragment are from a cyanobacterium Anabaena pre-tRNA-Leu gene, a Tetrahymena pre-rRNA, or a T4 phage td gene.
  • the 3′ half of Group I catalytic intron fragment and the 5’ Group I catalytic intron fragment are from a T4 phage td gene.
  • the 3′ exon fragment may include the first annealing region and the 5’ half of Group I catalytic intron fragment may include the second annealing region.
  • the first annealing region may include, e.g., from 2 to 16 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) ribonucleotides
  • the second annealing region may include, e.g., from 2 to 16 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) ribonucleotides.
  • the 3′ half of Group I catalytic intron fragment is the 5’ terminus of the linear polynucleotide.
  • the 5′ half of Group I catalytic intron fragment is the 3’ terminus of the linear polyribonucleotide.
  • the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- AACAACAGATAACTTACAGCTAGTCGGAAGGTGCAGAGACTCGACGGGAGCTACCC TAACGTCAAGACGAGGGTAAAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAG CGAAAGCTGCGGGAGAATG-3’ (SEQ ID NO: 172).
  • the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- AAATAATTGAGCCTTAGAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGA AACCTAAATCTAGCTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAG TAAGTT-3’ (SEQ ID NO: 173)).
  • the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 172 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 173.
  • the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- CTTCTGTTGATATGGATGCAGTTCACAGACTAAATGTCGGTCGGGGAAGATGTATTC TTCTCATAAGATATAGTCGGACCTCTCCTTAATGGGAGCTAGCGGATGAAGTGATGC AACACTGGAGCCGCTGGGAACTAATTTGTATGCGAAAGTATATTGATTAGTTTTGGA GTACTCG-3’ (SEQ ID NO: 174).
  • the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- AAATAGCAATATTTACCTTTGGAGGGAAAAGTTATCAGGCATGCACCTGGTAGCTAG TCTTTAAACCAATAGATTGCATCGGTTTAAAAGGCAAGACCGTCAAATTGCGGGAA AGGGGTCAACAGCCGTTCAGTACCAAGTCTCAGGGGAAACTTTGAGATGGCCTTGC AAAGGGTATGGTAATAAGCTGACGGACATGGTCCTAACCACGCAGCCAAGTCCTAA GTCAACAGAT-3’ (SEQ ID NO: 175).
  • the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 174 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 175.
  • the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- GGTTCTACATAAATGCCTAACGACTATCCCTTTGGGGAGTAGGGTCAAGTGACTCGA AACGATAGACAACTTGCTTTAACAAGTTGGAGATATAGTCTGCTCTGCATGGTGACA TGCAGCTGGATATAATTCCGGGGTAAGATTAACGACCTTATCTGAACATAATG-3’ (SEQ ID NO: 176).
  • the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- TAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACC TCTCTAGTAGACAATCCCGTGCTAAATTGTAGGACT-3’ (SEQ ID NO: 177).
  • the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 176 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 177.
  • the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- TAAACAACTAACAGCTTTAGAAGGTGCAGAGACTAGACGGGAGCTACCCTAACGGA TTCAGCCGAGGGTAAAGGGATAGTCCAATTCTCAACATCGCGATTGTTGATGGCAGC GAAAGTTGCAGAGAGAATGAAAATCCGCTGACTGTAAAGGTCGTGAGGGTTCGAGT CCCTCCGCCCCCA-3’ (SEQ ID NO: 178).
  • the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- ACGGTAGACGCAGCGGACTTAGAAAACTGGGCCTCGATCGCGAAAGGGATCGAGTG GCAGCTCTCAAACTCAGGGAAACCTAAAACTTTAAACATTMAAGTCATGGCAATCC TGAGCCAAGCTAAAGC-3’ (SEQ ID NO: 179).
  • the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 178 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 179.
  • the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- TTAAACTCAAAATTTAAAATCCCAAATTCAAAATTCCGGGAAGGTGCAGAGACTCG ACGGGAGCTACCCTAACGTAAAGCCGAGGGTAAAGGGAGAGTCCAATTCTCAAAGC LRN Ref. No.: LRN23-107WO Fish Ref.
  • the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- ATGGTAGACGCTACGGACTTAGAAAACTGAGCCTTGATAGAGAAATCTTTTAAGTG GAAGCTCTCAAATTCAGGGAAACCTAAATCTGAATACAGATATGGCAATCCTGAGC CAAGCCCAGAAAATTTAGACTTGAGATTTGATTTTGGAG-3’ (SEQ ID NO: 181).
  • the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 180 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 181.
  • the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- GGCTTTCAATTTGAAATCAGAAATTCAAAATTCAGGGAAGGTGCAGAGACTCGACG GGAGCTACCCTAACGTAAAGGCGAGGGTAAAGGGAGAGTCCAATTCTTAAAGCCTG AAGTTGTGCAAGCAACAAGGCAACAGTGAAAGCTGTGGAAGAATGAAAATCCGTTG ACCTTAAACGGTCGTGGGGGTTCAAGTCCCCCCACCCCC-3’ (SEQ ID NO: 182).
  • the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- ATGGTAGACGCTACGGACTTAGAAAACTGAGCCTTGATAGAGAAATCTTTCAAGTG GAAGCTCTCAAATTCAGGGAAACCTAAATCTGAATACAGATATGGCAATCCTGAGC CAAGCCCGGAAATTTTAGAATCAAGATTTTATTTT-3’ (SEQ ID NO: 183).
  • the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 182 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 183. In some embodiments, the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- AGAAATGGAGAAGGTGTAGAGACTGGAAGGCAGGCACCCTAACGTTAAAGGCGAG GGTGAAGGGACAGTCCAGACCACAAACCAGTAAATCTGGGCAGCGAAAGCTGTAGA TGGTAAGCATAACCCGAAGGTCAGTGGTTCAAATCCACTTCCCGCCACCAAATTAAA AAAACAATAA-3’ (SEQ ID NO: 184).
  • the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- AGAAATGGAGAAGGTGTAGAGACTGGAAGGCAGGCACCCTAACGTTAAAGGCGAG GGTGAAGGGACAGTCCAGACCACAAACCAGTAAATCTGGGCAGCGAAAGCTGTAGA TGGTAAGCATAACCCGAAGGTCAGTGGTTCAAATCCACTTCCCGCCACCAAATTAAA AAAACAATAA-3’ (SEQ ID NO: 185).
  • the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 184 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 185. In some embodiments, the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- ACAACAGATAACTTACTAACTTACAGCTAGTCGGAAGGTGCAGAGACTCGACGGGA GCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAG GCAGTAGCGAAAGCTGCGGGAGAATGAAAATCCGTAGCGTCTAAACGGTCGTGTGG GTTCAAGTCCCTCCACCCCCA-3’ (SEQ ID NO: 186).
  • the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- AGACGCTACGGACTTAAATAATTGAGCCTTAGAGAAGAAATTCTTTAAGTGGATGCT CTCAAACTCAGGGAAACCTAAATCTAGCTATAGACAAGGCAATCCTGAGCCAAGCC GAAGTAGTAATTAGTAAGTTAGTAAGTT-3’ (SEQ ID NO: 187).
  • the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 186 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 187. In some embodiments, the 3’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- AACAACAGATAACTTACTAGTTACTAGTCGGAAGGTGCAGAGACTCGACGGGAGCT ACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCA GTAGCGAAAGCTGCGGGAGAATGAAAATCCGTAGCGTCTAAACGGTCGTGTGGGTT CAAGTCCCTCCACCCCCA-3’ (SEQ ID NO: 188).
  • the 5’ half of Group I catalytic intron fragment has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of 5’- LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 AGACGCTACGGACTTAAATAATTGAGCCTTAGAGAAGAAATTCTTTAAGTGGATGCT CTCAAACTCAGGGAAACCTAAATCTAGCTATAGACAAGGCAATCCTGAGCCAAGCC GAAGTAGTAATTAGTAAGTT-3’ (SEQ ID NO: 189).
  • the 3’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 188 and the 5’ half of Group I catalytic intron fragment has the sequence of SEQ ID NO: 189.
  • the Group I catalytic intron fragment is from the T4 phage nrdB gene or nrdD gene.
  • the 3′ half of Group I catalytic intron fragment of includes a sequence having at least 80% sequence identity to 5’- TTGCAAAACAAGGTTCAACGACTAGTCTTCGGACGTAGGGTCAAGCGACTCGAAAT GGGGAGAATCCCTCCGGGATTGTGATATAGTCTGGACTGCATGGTAACATGCAGCA GTTCATAAGAGAACGGGTTGAGAATTAGCGAGCTCAATCGAACATACG-3’ (SEQ ID NO: 190).
  • the 3′ half of Group I catalytic intron fragment of (A) includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to 5’- TTGCAAAACAAGGTTCAACGACTAGTCTTCGGACGTAGGGTCAAGCGACTCGAAAT GGGGAGAATCCCTCCGGGATTGTGATATAGTCTGGACTGCATGGTAACATGCAGCA GTTCATAAGAGAACGGGTTGAGAATTAGCGAGCTCAATCGAACATACG-3’ (SEQ ID NO: 191).
  • the 5′ half of Group I catalytic intron fragment from the T4 phage nrdB gene includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to 5’- TTGCAAAACAAGGTTCAACGACTAGTCTTCGGACGTAGGGTCAAGCGACTCGAAAT GGGGAGAATCCCTCCGGGATTGTGATATAGTCTGGACTGCATGGTAACATGCAGCA GTTCATAAGAGAACGGG
  • the 3′ half of Group I catalytic intron fragment is from the T4 phage nrdB gene and the 5′ half of Group I catalytic intron fragment is from the T4 phage nrdB gene.
  • the 5′ half of Group I catalytic intron includes a sequence having at least 80% sequence identity to 5’- AAAATGCGCCTTTAAACGGTAACGTTTATCGAAAACTCCTTTAATTGCTGGAAAGTC CTTTATGGAAAACTAGCAGCCAAGGTTTTGCTT-3’ (SEQ ID NO: 192).
  • the 5′ half of Group I catalytic intron includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to 5’- AAAATGCGCCTTTAAACGGTAACGTTTATCGAAAACTCCTTTAATTGCTGGAAAGTC CTTTATGGAAAACTAGCAGCCAAGGTTTTGCTT-3’ (SEQ ID NO: 193).
  • the 3′ half of Group I catalytic intron fragment is from the T4 phage nrdD gene.
  • the 3′ half of Group I catalytic intron fragment includes a sequence having at least 80% sequence identity to 5’- CAGTAGCTGTAAATGCCCAACGACTATCCCTGATGAATGTAAGGGAGTAGGGTCAA GCGACCCGAAACGGCAGACAACTCTAAGAGTTGAAGATATAGTCTGAACTGCATGG TGACATGCAGCTGTTTATCCTCGTATAAATATGAATACGAGGTGAAACGATGAAATG AATTACATTGTTTCATATAAACGGGTAGAGAAGTAGCGAACTCTACTGAACACATTG -3’ (SEQ ID NO: 194).
  • the 3′ half of Group I catalytic intron fragment includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to 5’- CAGTAGCTGTAAATGCCCAACGACTATCCCTGATGAATGTAAGGGAGTAGGGTCAA GCGACCCGAAACGGCAGACAACTCTAAGAGTTGAAGATATAGTCTGAACTGCATGG TGACATGCAGCTGTTTATCCTCGTATAAATATGAATACGAGGTGAAACGATGAAATG AATTACATTGTTTCATATAAACGGGTAGAGAAGTAGCGAACTCTACTGAACACATTG -3’ (SEQ ID NO: 195).
  • the 5′ half of Group I catalytic intron fragment is from the T4 phage nrdD gene.
  • the 3′ half of Group I catalytic intron fragment is from the T4 phage nrdD gene and the 5′ half of Group I catalytic intron fragment is from the T4 phage nrdD gene.
  • the 5′ half of Group I catalytic intron includes a sequence having at least 80% sequence identity to 5’- TAACGTAAGTCAAGCTCATGTAAAATCTGCCTAAAACGGGAAACTCTCACTGAGAC AATCCGTTGCTAAATCAG-3’ (SEQ ID NO: 196).
  • the 5′ half of Group I catalytic intron includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to 5’- TAACGTAAGTCAAGCTCATGTAAAATCTGCCTAAAACGGGAAACTCTCACTGAGAC AATCCGTTGCTAAATCAG-3’ (SEQ ID NO: 197).
  • the 3’ exon fragment includes a sequence having at least 80% sequence identity to 5’- LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 GTACCTTTAACTTCCATAAGAACATGGAAATCATGGAAGGTAATGCCAAG-3’ (SEQ ID NO: 198).
  • the 3’ exon fragment includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to 5’- GTACCTTTAACTTCCATAAGAACATGGAAATCATGGAAGGTAATGCCAAG-3’ (SEQ ID NO: 199). In some embodiments, the 3’ exon fragment includes a sequence having at least 80% sequence identity to 5’- GTACCTTTAACTTCCAAAAGATACATAAAAATCATGGAAGGTAATGCCAAG-3’ (SEQ ID NO: 200.
  • the 3’ exon fragment includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to 5’- GTACCTTTAACTTCCAAAAGATACATAAAAATCATGGAAGGTAATGCCAAG-3’ (SEQ ID NO: 201).
  • the 5’ exon fragment includes a sequence having at least 80% sequence identity to 5’-TTTTTATGTATCTTTTGCGT-3’ (SEQ ID NO: 202).
  • the 5’ exon fragment includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to 5’-TTTTTATGTATCTTTTGCGT-3’ (SEQ ID NO: 203).
  • the 3’ exon fragment Includes a sequence having at least 80% sequence identity to 5’- ATGAAGTGAACACGTTATTCAGTTCAAACGGACAGACTCCTTTTGTAACA -3’ (SEQ ID NO: 204). In some embodiments, the 3’ exon fragment includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to 5’- ATGAAGTGAACACGTTATTCAGTTCAAACGGACAGACTCCTTTTGTAACA -3’ (SEQ ID NO: 205).
  • the 3’ exon fragment includes a sequence having at least 80% sequence identity to 5’- ATGAAGTGAACACGTTACATAAGCTTGGAATGCAGACTCCTTTTGTAACA -3’ (SEQ ID NO: 206).
  • the 3’ exon fragment includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to 5’- ATGAAGTGAACACGTTACATAAGCTTGGAATGCAGACTCCTTTTGTAACA -3’ (SEQ ID NO: 207).
  • the 5’ exon fragment includes a sequence having at least 80% sequence identity to 5’- TGCATTCCAAGCTTATGAGT -3’ (SEQ ID NO: 208). In some embodiments, the 5’ exon fragment includes a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to 5’- TGCATTCCAAGCTTATGAGT -3’ (SEQ ID NO: 209).
  • a linear polyribonucleotide for circularization may be cyclized or concatenated by a non-nucleic acid moiety that causes an attraction between atoms, molecular surfaces at, near, or linked to the 5’ and 3’ ends of the linear polyribonucleotide for circularization.
  • one or more linear polyribonucleotides is cyclized or concatenated by intermolecular forces or intramolecular forces.
  • intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole- induced dipole forces, Van der Waals forces, and London dispersion forces.
  • intramolecular forces include covalent bonds, metallic bonds, ionic bonds, resonant bonds, agnostic bonds, dipolar bonds, conjugation, hyperconjugation and antibonding.
  • a linear polyribonucleotide for circularization may include a ribozyme RNA sequence near the 5’ terminus and near the 3’ terminus.
  • the ribozyme RNA sequence may covalently link to a peptide when the sequence is exposed to the remainder of the ribozyme.
  • the peptides covalently linked to the ribozyme RNA sequence near the 5’ terminus and the 3 ‘terminus may associate with each other causing a linear polyribonucleotide to cyclize or concatenate.
  • the peptides covalently linked to the ribozyme RNA near the 5’ terminus and the 3’ terminus may cause the linear primary construct or linear mRNA to cyclize or concatenate after being subjected to ligation using various methods known in the art such as, but not limited to, protein ligation.
  • Non-limiting examples of ribozymes for use in the linear primary constructs or linear polyribonucleotides of the present invention or a non-exhaustive listing of methods to incorporate or covalently link peptides are described in U.S. Patent Publication No. U.S. 2003/0082768, the contents of which is here in incorporated by reference in its entirety.
  • chemical methods of circularization may be used to generate the circular polyribonucleotide.
  • Such methods may include but are not limited to click chemistry (e.g., alkyne and azide-based methods, or clickable bases), olefin metathesis, phosphoramidate ligation, hemiaminal-imine crosslinking, base modification, and any combination thereof.
  • click chemistry e.g., alkyne and azide-based methods, or clickable bases
  • olefin metathesis e.g., alkyne and azide-based methods, or clickable bases
  • phosphoramidate ligation e.g., phosphoramidate ligation
  • hemiaminal-imine crosslinking hemiaminal-imine crosslinking
  • base modification e.g., base modification, and any combination thereof.
  • the 5’-end and the 3’-end of a linear polyribonucleotide for circularization includes chemically reactive groups that, when close together, may form a new covalent linkage between the 5’-end and the 3’-end of
  • the 5’-end may contain an NHS-ester reactive group and the 3’-end may contain a 3’-amino-terminated nucleotide such that in an organic solvent the 3’-amino-terminated nucleotide on the 3’-end of a linear RNA molecule will undergo a nucleophilic attack on the 5’-NHS-ester moiety forming a new 5’-/3’-amide bond.
  • the circular polyribonucleotide may be produced using a deoxyribonucleotide template transcribed in a cell-free system (e.g., by in vitro transcription) to a produce a linear RNA.
  • the linear polyribonucleotide produces a splicing-compatible polyribonucleotide, which may be self-spliced to produce a circular polyribonucleotide.
  • the disclosure provides a method of producing a circular polyribonucleotide (e.g., in a cell-free system) by providing a linear polyribonucleotide; and self- splicing linear polyribonucleotide under conditions suitable for splicing of the 3’ and 5’ splice sites of the linear polyribonucleotide; thereby producing a circular polyribonucleotide.
  • the disclosure provides a method of producing a circular polyribonucleotide by providing a deoxyribonucleotide encoding the linear polyribonucleotide; transcribing the deoxyribonucleotide in a cell-free system to produce the linear polyribonucleotide; optionally purifying the splicing-compatible linear polyribonucleotide; and self-splicing the linear polyribonucleotide under conditions suitable for splicing of the 3’ and 5’ splice sites of the linear polyribonucleotide, thereby producing a circular polyribonucleotide.
  • the disclosure provides a method of producing a circular polyribonucleotide by providing a deoxyribonucleotide encoding a linear polyribonucleotide; transcribing the deoxyribonucleotide in a cell-free system to produce the linear polyribonucleotide, wherein the transcribing occurs in a solution under conditions suitable for splicing of the 3’ and 5’ splice sites of the linear polyribonucleotide, thereby producing a circular polyribonucleotide.
  • the linear polyribonucleotide comprises a 5’ split- intron and a 3’ split-intron (e.g., a self-splicing construct for producing a circular LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 polyribonucleotide).
  • the linear polyribonucleotide comprises a 5’ annealing region and a 3’ annealing region.
  • Suitable conditions for in vitro transcriptions and or self-splicing may include any conditions (e.g., a solution or a buffer, such as an aqueous buffer or solution) that mimic physiological conditions in one or more respects.
  • suitable conditions include between 0.1-100mM Mg2+ ions or a salt thereof (e.g., 1-100mM, 1-50mM, 1-20mM, 5- 50mM, 5-20 mM, or 5-15mM). In some embodiments, suitable conditions include between 1- 1000mM K+ ions or a salt thereof such as KCl (e.g., 1-1000mM, 1-500mM, 1-200mM, 50- 500mM, 100-500mM, or 100-300mM).
  • KCl e.g., 1-1000mM, 1-500mM, 1-200mM, 50- 500mM, 100-500mM, or 100-300mM.
  • suitable conditions include between 1-1000mM Cl- ions or a salt thereof such as KCl (e.g., 1-1000mM, 1-500mM, 1- 200mM, 50- 500mM, 100-500mM, or 100-300mM).
  • suitable conditions include between 0.1-100mM Mn2+ ions or a salt thereof such as MnCl2 (e.g., 0.1-100mM, 0.1- 50mM, 0.1-20mM, 0.1-10mM, 0.1-5mM, 0.1-2mM, 0.5- 50mM, 0.5-20 mM, 0.5-15mM, 0.5- 5mM, 0.5-2mM, or 0.1-10mM).
  • suitable conditions include dithiothreitol (DTT) (e.g., 1-1000 ⁇ M, 1-500 ⁇ M, 1-200 ⁇ M, 50- 500 ⁇ M, 100-500 ⁇ M, 100-300 ⁇ M, 0.1- 100mM, 0.1-50mM, 0.1-20mM, 0.1-10mM, 0.1-5mM, 0.1-2mM, 0.5- 50mM, 0.5-20 mM, 0.5- 15mM, 0.5-5mM, 0.5-2mM, or 0.1-10mM).
  • DTT dithiothreitol
  • suitable conditions include between 0.1mM and 100mM ribonucleoside triphosphate (NTP) (e.g., 0.1-100 mM, 0.1-50mM, 0.1-10mM, 1- 100mM, 1-50mM, or 1-10mM).
  • NTP ribonucleoside triphosphate
  • suitable conditions include a pH of 4 to 10 (e.g., pH of 5 to 9, pH of 6 to 9, or pH of 6.5 to 8.5).
  • suitable conditions include a temperature of 4°C to 50°C (e.g., 10°C to 40°C, 15 °C to 40°C, 20°C to 40°C, or 30°C to 40°C),
  • the linear polyribonucleotide is produced from a deoxyribonucleic acid, e.g., a deoxyribonucleic acid described herein, such as a DNA vector, a linearized DNA vector, or a cDNA.
  • the linear polyribonucleotide is transcribed from the deoxyribonucleic acid by transcription in a cell-free system (e.g., in vitro transcription).
  • the circular polyribonucleotide may be produced in a cell, e.g., a prokaryotic cell or a eukaryotic cell.
  • an exogenous polyribonucleotide is provided to a cell (e.g., a linear polyribonucleotide described herein or a DNA molecule encoding for the transcription of a linear polyribonucleotide described here).
  • the linear polyribonucleotides may be transcribed in the cell from an exogenous DNA molecule provided to the cell.
  • polyribonucleotide may be transcribed in the cell from an exogenous recombinant DNA molecule transiently provided to the cell.
  • the exogenous DNA molecule does not integrate into the cell’s genome.
  • the linear polyribonucleotide is transcribed in the cell from a recombinant DNA molecule that is incorporated into the cell’s genome.
  • the cell is a prokaryotic cell.
  • the prokaryotic cell including the polyribonucleotides described herein is a bacterial cell or an archaeal cell.
  • the prokaryotic cell including the polyribonucleotides described herein may be E coli, halophilic archaea (e.g., Haloferax volcaniii), Sphingomonas, cyanobacteria (e.g., Synechococcus elongatus, Spirulina (Arthrospira) spp., and Synechocystis spp.), Streptomyces, actinomycetes (e.g., Nonomuraea, Kitasatospora, or Thermobifida), Bacillus spp.
  • E coli halophilic archaea
  • Sphingomonas e.g., cyanobacteria (e.g., Synechococcus elongatus, Spirulina (Arthrospira) spp., and Synechocystis spp.)
  • Streptomyces e.g., Nonom
  • the prokaryotic cells may be grown in a culture medium.
  • the prokaryotic cells may be contained in a bioreactor.
  • the cell is a eukaryotic cell.
  • the eukaryotic cell is a unicellular eukaryotic cell.
  • the unicellular eukaryotic is a unicellular fungal cell such as a yeast cell (e.g., Saccharomyces cerevisiae and other Saccharomyces spp., Brettanomyces spp., Schizosaccharomyces spp., Torulaspora spp, and Pichia spp.).
  • the unicellular eukaryotic cell is a unicellular animal cell.
  • a unicellular animal cell may be a cell isolated from a multicellular animal and grown in culture, or the daughter cells thereof.
  • the unicellular animal cell is dedifferentiated.
  • the unicellular eukaryotic cell is a unicellular plant cell.
  • a unicellular plant cell may be a cell isolated from a multicellular plant and grown in culture, or the daughter cells thereof. In some embodiments, the unicellular plant cell is dedifferentiated. In some embodiments, the unicellular plant cell is from a plant callus. In embodiments, the unicellular cell is a plant cell protoplast. In some embodiments, the unicellular eukaryotic cell is a unicellular eukaryotic algal cell, such as a unicellular green alga, a diatom, an euglenid, or a dinoflagellate.
  • Non-limiting examples of unicellular eukaryotic algae of interest include Dunaliella salina, Chlorella vulgaris, Chlorella zofingiensis, Haematococcus pluvialis, Neochloris oleoabundans and other Neochloris spp., Protosiphon botryoides, Botryococcus LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 braunii, Cryptococcus spp., Chlamydomonas reinhardtii and other Chlamydomonas spp.
  • the unicellular eukaryotic cell is a protist cell.
  • the unicellular eukaryotic cell is a protozoan cell.
  • the eukaryotic cell is a cell of a multicellular eukaryote.
  • the multicellular eukaryote may be selected from the group consisting of a vertebrate animal, an invertebrate animal, a multicellular fungus, a multicellular alga, and a multicellular plant.
  • the eukaryotic organism is a human.
  • the eukaryotic organism is a non-human vertebrate animal.
  • the eukaryotic organism is an invertebrate animal.
  • the eukaryotic organism is a multicellular fungus. In some embodiments, the eukaryotic organism is a multicellular plant. In embodiments, the eukaryotic cell is a cell of a human or a cell of a non-human mammal such as a non-human primate (e.g., monkeys, apes), ungulate (e.g., bovids including cattle, buffalo, bison, sheep, goat, and musk ox; pig; camelids including camel, llama, and alpaca; deer, antelope; and equids including horse and donkey), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse, guinea pig, hamster, squirrel), or lagomorph (e.g., rabbit, hare).
  • a non-human primate e.g., monkeys, apes
  • ungulate e.g., bovids
  • the eukaryotic cell is a cell of a bird, such as a member of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots).
  • avian taxa Galliformes e.g., chickens, turkeys, pheasants, quail
  • Anseriformes e.g., ducks, geese
  • Paleaognathae e.g., ostriches, emus
  • Columbiformes e.g., pigeons, doves
  • the eukaryotic cell is a cell of an arthropod (e.g., insects, arachnids, crustaceans), a nematode, an annelid, a helminth, or a mollusc.
  • the eukaryotic cell is a cell of a multicellular plant, such as an angiosperm plant (which can be a dicot or a monocot) or a gymnosperm plant (e.g., a conifer, a cycad, a gnetophyte, a Ginkgo), a fern, horsetail, clubmoss, or a bryophyte.
  • the eukaryotic cell is a cell of a eukaryotic multicellular alga.
  • the eukaryotic cells may be grown in a culture medium.
  • the eukaryotic cells may be contained in a bioreactor.
  • any method of producing a circular polyribonucleotide described herein may be performed in a bioreactor.
  • a bioreactor refers to any vessel in which a chemical or biological process is carried out which involves organisms or biochemically active substances derived from such organisms. Bioreactors may be compatible with the cell-free methods for production of circular RNA described herein.
  • a vessel for a bioreactor may include a culture flask, a dish, or a bag that may be single use (disposable), autoclavable, or sterilizable.
  • a LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 bioreactor may be made of glass, or it may be polymer-based, or it may be made of other materials.
  • bioreactors include, without limitation, stirred tank (e.g., well mixed) bioreactors and tubular (e.g., plug flow) bioreactors, airlift bioreactors, membrane stirred tanks, spin filter stirred tanks, vibromixers, fluidized bed reactors, and membrane bioreactors.
  • the mode of operating the bioreactor may be a batch or continuous processes.
  • a bioreactor is continuous when the reagent and product streams are continuously being fed and withdrawn from the system.
  • a batch bioreactor may have a continuous recirculating flow, but no continuous feeding of reagents or product harvest.
  • Some methods of the present disclosure are directed to large-scale production of circular polyribonucleotides.
  • the method may be performed in a volume of 1 liter (L) to 50 L, or more (e.g., 5 L, 10 L, 15 L, 20 L, 25 L, 30 L, 35 L, 40 L, 45 L, 50 L, or more).
  • the method may be performed in a volume of 5 L to 10 L, 5 L to 15 L, 5 L to 20 L, 5 L to 25 L, 5 L to 30 L, 5 L to 35 L, 5 L to 40 L, 5 L to 45 L, 10 L to 15 L, 10 L to 20 L, 10 L to 25 L, 20 L to 30 L, 10 L to 35 L, 10 L to 40 L, 10 L to 45 L, 10 L to 50 L, 15 L to 20 L, 15 L to 25 L, 15 L to 30 L, 15 L to 35 L, 15 L to 40 L, 15 L to 45 L, or 15 to 50 L.
  • a bioreactor may produce at least 1g of circular RNA.
  • a bioreactor may produce 1-200g of circular RNA (e.g., 1-10g, 1-20g, 1-50g, 10-50g, 10-100g, 50-100g, or 50-200g of circular RNA).
  • the amount produced is measured per liter (e.g., 1-200g per liter), per batch or reaction (e.g., 1-200g per batch or reaction), or per unit time (e.g., 1-200g per hour or per day).
  • more than one bioreactor may be utilized in series to increase the production capacity (e.g., one, two, three, four, five, six, seven, eight, or nine bioreactors may be used in series).
  • circularization efficiency is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100%. In some embodiments, circularization efficiency is at least about 40%. In some embodiments, circularization efficiency is between about 10% and about 100%; for example, circularization efficiency is about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, LRN Ref.
  • circularization efficiency is between about 20% and about 80%. In some embodiments, circularization efficiency is between about 30% and about 60%. In some embodiments, circularization efficiency is about 40%.
  • the linear polyribonucleotide is substantively enriched or pure (e.g., purified) prior to self-splicing the linear polyribonucleotide. In other embodiments, the linear polyribonucleotide is not purified prior to self-splicing the linear polyribonucleotide.
  • the resulting circular polyribonucleotide is purified.
  • Purification may include separating or enriching the desired reaction product from one or more undesired components, such as any unreacted stating material, byproducts, enzymes, or other reaction components.
  • purification of linear polyribonucleotide following LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 transcription in a cell-free system may include separation or enrichment from the DNA template prior to self-splicing the linear polyribonucleotide.
  • Purification of the circular RNA product following splicing may be used to separate or enrich the circular polyribonucleotide from its corresponding linear polyribonucleotide.
  • Methods of purification of polyribonucleotides are known to those of skill in the art and include enzymatic purification or by chromatography. In some embodiments, the methods of purification result in a circular polyribonucleotide that has less than 50% (e.g., less than 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1%) linear polyribonucleotides.
  • the reference criterion for the amount of linear polyribonucleotide molecules present in a preparation is the presence of no more than 1 ng/ml, 5 ng/ml, 10 ng/ml, 15 ng/ml, 20 ng/ml, 25 ng/ml, 30 ng/ml, 35 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 1 ⁇ g/ ml, 10 ⁇ g/ml, 50 ⁇ g/ml, 100 ⁇ g/ml, 200 g/ml, 300 ⁇ g/ml, 400 ⁇ g/ml, 500 ⁇ g/ml, 600 ⁇ g/ml, 600 ⁇ g/ml, 500
  • the reference criterion for the amount of circular polyribonucleotide molecules present in a preparation is at least 30% (w/w), 40% (w/w), 50% (w/w), 60% (w/w), 70% (w/w), 80% (w/w), 85% (w/w), 90% (w/w), 91% (w/w), 92% (w/w), 93% (w/w), 94% (w/w), 95% (w/w), 96% (w/w), 97% (w/w), 98% (w/w), 99% (w/w), 99.1% (w/w), 99.2% (w/w), 99.3% (w/w), 99.4% (w/w), 99.5% (w/w), 99.6% (w/w), 99.7% (w/w), 99.8% (w/w), 99.9% (w/w), or 100% (w/w) molecules of the total ribonucleotide molecules in the preparation.
  • the reference criterion for the amount of linear polyribonucleotide molecules present in a preparation is no more than 0.5% (w/w), 1% (w/w), 2% (w/w), 5% (w/w), 10% (w/w), 15% (w/w), 20% (w/w), 25% (w/w), 30% (w/w), 40% (w/w), 50% (w/w) linear polyribonucleotide molecules of the total ribonucleotide molecules in the preparation.
  • the reference criterion for the amount of nicked polyribonucleotide molecules present in a preparation is no LRN Ref.
  • the reference criterion for the amount of combined nicked and linear polyribonucleotide molecules present in a preparation is no more than 0.5% (w/w), 1% (w/w), 2% (w/w), 5% (w/w), 10% (w/w), 15% (w/w), 20% (w/w), 25% (w/w), 30% (w/w), 40% (w/w), 50% (w/w) combined nicked and linear polyribonucleotide molecules of the total ribonucleotide molecules in the preparation.
  • a preparation e.g., pharmaceutical preparation
  • a preparation is an intermediate preparation of a final circular polyribonucleotide drug product.
  • a preparation is a drug substance or active pharmaceutical ingredient (API).
  • a preparation is a drug product for administration to a subject.
  • a preparation e.g., pharmaceutical preparation
  • a preparation (e.g., pharmaceutical preparation) of circular polyribonucleotides is (before, during or after the reduction of linear polyribonucleotide) further processed to substantially remove DNA, protein contamination (e.g., cell protein such as a host cell protein or protein process impurities), endotoxin, mononucleotide molecules, and/or a process-related impurity.
  • compositions and pharmaceutical compositions comprising (i) any of the circular polyribonucleotides or modified RNAs described herein and (ii) a pharmaceutically acceptable excipient.
  • Pharmaceutical compositions can comprise one or more additional therapeutic agents, e.g., therapeutically and/or prophylactically active agents.
  • additional therapeutic agents e.g., therapeutically and/or prophylactically active agents.
  • Methods of making pharmaceutical compositions including circular polyribonucleotides or modified RNAs are described in WO 2020/181013, which is incorporated herein by reference in its entirety.
  • Pharmaceutical compositions provided herein are suitable for administration to humans or any other animal, e.g., to non-human animals, e.g., non-human mammals.
  • compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood.
  • subjects that can be administered any of the pharmaceutical compositions described herein include humans, non-human primate, mammals (e.g., commercially-relevant mammals, LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 such as cattle, pigs, horses, sheep, cats, dogs, mice, and rats), and birds (e.g., including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys).
  • Pharmaceutical compositions provided herein can be formulated for any suitable mode of administration.
  • Non-limiting examples include formulation of the pharmaceutical composition for intravenous, subcutaneous, intrahepatic, or intramuscular administration.
  • a pharmaceutical composition is formulated for local administration.
  • a pharmaceutical composition is formulated for systemic administration.
  • Pharmaceutical compositions provided herein can include an unencapsulated circular polyribonucleotide of modified RNA, a partially encapsulated circular polyribonucleotide or modified RNA, or a completely encapsulated circular polyribonucleotide or modified RNA. Any of the circular polyribonucleotides or modified RNAs described herein can be included in a pharmaceutical composition with a carrier or without a carrier.
  • compositions described herein can be formulated with a carrier, e.g., pharmaceutical carrier and/or a polymeric carrier, e.g., a liposome.
  • a carrier e.g., pharmaceutical carrier and/or a polymeric carrier, e.g., a liposome.
  • the circular polyribonucleotide or modified RNA can be formulated as a lipid nanoparticle.
  • the circular polyribonucleotide or modified RNA is formulated for unencapsulated delivery or natural nanoparticle delivery.
  • the natural nanoparticle is composed of a natural source selected from a plant, bacteria, animal, insect, archaea, or fungi.
  • the circular polyribonucleotide is formulated within (a) a plurality of lipid nanoparticles (LNP) comprising synthetic structural lipids and an ionizable lipid or within (b) a natural nanoparticle comprising natural lipids and an ionizable lipid.
  • LNP lipid nanoparticles
  • One or more naturally occurring and/or synthetic lipid compounds may be used in the preparation of a lipid-based carrier (or lipid nanoformulation).
  • a lipid-based carrier (or lipid nanoformulation) may be a lipid particle that has a complexity characterized by comprising a wide variety of lipids, including both synthetic lipids or lipids extracted from one or more natural sources (such as plants or bacteria).
  • Natural lipids can be derived from a variety of arthropod, fungi, plant, archaea, or bacteria, or one or more parts thereof (e.g., segments, organs, eggs, spores, mycelium, tissue, membrane or cell wall).
  • the lipid-based carrier is a modified natural nanoparticle.
  • modified natural particle refers to a natural lipid LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 nanoparticles composition including both naturally derived lipids and one or more synthetically derived lipids.
  • modified natural nanoparticles may include ionizable lipids, non-cationic lipids, sterols, and PEGylated lipids. More embodiments of natural lipids, complex lipid particles, and methods of producing and delivering natural nanoparticles may be found in PCT Application No. PCT/US2023/021094, filed on May 5, 2023, PCT Application No. PCT/US24/25134, filed on April 18, 2024, and PCT Application No. PCT/US24/25270, filed on April 18, 2024, the contents of which are incorporated herein by reference in their entirety.
  • the modified natural nanoparticle is plant-based, e.g. as described in International Patent Publication Nos.
  • the modified natural nanoparticle is bacteria-derived, e.g. as described in International Patent Publication Nos. WO2023/096858.
  • more than one ionizable lipid can be used for the ionizable lipid component. More embodiments of ionizable lipids may be found in PCT Application No. PCT/US22/50725, filed on November 22, 2022, PCT Application No.
  • Suitable lipids for use in the RNA composition and methods for making and using thereof include the lipids as described in J. McClellan, M. C. King, Cell 2010, 141, 210-217 and in Whitehead et al., Nature Communications (2014) 5:4277, which is incorporated herein by reference in its entirety.
  • the ionizable lipid may be 1,1’-((2-(4-(2-((2-(bis(2- hydroxydodecyl)amino)ethyl) (2-hydroxydodecyl)amino)ethyl)piperazin-1- yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LP01, 5A2-SC8, Lipid 5, SM-102 (Lipid H), or ALC-315.
  • the ionizable lipid included is LP01.
  • the natural nanoparticle may include one or more exogenous lipids, e.g., lipids that are exogenous to the natural source (e.g., originating from a source that is not the source or source part from which the particle is produced).
  • the lipid composition may include 0%, less than 1%, or at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% exogenous lipid.
  • the exogenous lipid e.g., ionizable lipid
  • the exogenous lipid is added to the preparation prior to step (b), e.g., mixed with extracted natural lipids prior to step (b).
  • the term “exogenous lipid” refers to a lipid that is exogenous to the natural source (e.g., plant, bacteria), i.e., a lipid originates from a source that is not the natural source from which the lipids are extracted (e.g., a lipid that is added to the lipid particle formulation using method described herein).
  • the term “exogenous lipid” does not exclude a natural-derived lipid (such as a plant-derived sterol).
  • an exogenous lipid can be a natural-derived lipid (such as a plant-derived sterol that is exogenous to the plant source from which the lipids are extracted, e.g., an exogenous lipid can be a plant derived sterol that is added to the lipid particle formulation).
  • an exogenous lipid can be a natural- derived lipid that is exogenous to the particular natural source from which the lipids are extracted (e.g., a bacteria-derived lipid that is exogenous to the plant source from which the lipids are extracted, or vice versa).
  • An exogenous lipid may be a cell-penetrating agent, may be capable of increasing delivery of one or more polynucleotides by the lipid formulation (e.g. lipid nanoparticle or natural nanoparticle) to a cell, and/or may be capable of increasing loading (e.g., loading efficiency or loading capacity) of a polynucleotide.
  • the exogenous lipid may be a stabilizing lipid.
  • the exogenous lipid may be a structural lipid (e.g., a synthetic structural lipid).
  • Exemplary exogenous lipids include ionizable lipids, synthetic structural lipids, sterols, and PEGylated lipids.
  • Exogenous lipids may also include cationic lipids.
  • cationic lipid refers to an amphiphilic LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 molecule (e.g., a lipid or a lipidoid) that is positively charged, containing a cationic group (e.g., a cationic head group).
  • the term “ionizable lipid” refers to an amphiphilic molecule (e.g., a lipid or a lipidoid, e.g., a synthetic lipid or lipidoid) containing a group (e.g., a head group) that can be ionized, e.g., dissociated to produce one or more electrically charged species, under a given condition (e.g., pH).
  • the natural nanoparticle may contain 3-1000 lipids extracted from one or more natural (e.g., plant, bacteria) sources.
  • the natural nanoparticle contains at least 10 natural lipids belonging to one or more of the classes selected from the group consisting of fatty acyls (FA), fatty acyl conjugates, phospholipids, glycerolipids, glycolipids, glycerophospholipids, sphingolipids, waxes, and sterol.
  • the lipid nanoparticle or natural nanoparticle composition comprises one or more ionizable lipids, one or more structural lipids (synthetic or natural), a sterol, and one or more PEG-modified lipids.
  • the natural nanoparticle may further comprise a neutral lipid as a helper lipid.
  • the natural lipids may be used in combination with a neutral lipid as a structural lipid component.
  • the particle has a size of less than about 200 nm.
  • the RNA and particle composition has an N:P ratio of at least 3, for instance, an N:P ratio of 3 to 100, 3 to 50, 3 to 30, 3 to 20, 3 to 15, 3 to 12, 6 to 30, 6 to 20, 6 to 15, or 6 to 12.
  • pharmaceutical compositions described herein can be formulated without a carrier, e.g., in a naked delivery formulation.
  • a naked delivery formulation refers to a formulation that is free from a carrier.
  • a naked delivery formulation can be free of any or all of: transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers.
  • a naked delivery formulation can be free from phtoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin, lipofectamine, polyethylenimine, poly(trimethylenimine), poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, l,2-Dioleoyl-3-Trimethylammonium- LRN Re
  • the naked delivery formulation can comprise a non-carrier excipient.
  • a non-carrier excipient may comprise an inactive ingredient that does not exhibit an active cell-penetrating effect.
  • a non-carrier excipient may comprise a buffer, for example PBS.
  • a non-carrier excipient may be a solvent, a non-aqueous solvent, a diluent, a suspension aid, a surface active agent, an isotonic agent, a thickening agent, an emulsifying agent, a preservative, a polymer, a peptide, a protein, a cell, a hyaluronidase, a dispersing agent, a granulating agent, a disintegrating agent, a binding agent, a buffering agent, a lubricating agent, or an oil.
  • the naked delivery formulation can comprise a diluent (e.g., a parenterally acceptable diluent).
  • a diluent can be a liquid diluent or a solid diluent.
  • a diluent can be an RNA solubilizing agent, a buffer, or an isotonic agent.
  • Non- limiting examples of an RNA solubilizing agent include water, ethanol, methanol, acetone, formamide, and 2-propanol.
  • Examples of a buffer include 2-(N-morpholino)ethanesulfonic acid (MES), Bis-Tris, 2-[(2-amino-2-oxoethyl)-(carboxymethyl)amino]acetic acid (ADA), N-(2- Acetamido)-2-aminoethanesulfonic acid (ACES), piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), 3- (N-morpholino)propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Tris, Tricine, Gly-Gly, Bicine, or phosphate.
  • MES 2-(N-morpholino)ethanesulfonic acid
  • Bis-Tris 2-[(2-amino-2
  • Non-limiting examples of an isotonic agent include glycerin, mannitol, polyethylene glycol, propylene glycol, trehalose, or sucrose.
  • Purity of the pharmaceutical composition comprising the circular polyribonucleotide or modified RNA can be measured using any method known in the art, e.g., chromatography, LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 electrophoresis, mass spectrometry, fluorescence, light scattering, refractive index, microscopy, circular dichroism (CD) spectroscopy, spectrophotometry, or surface plasmon resonance (SPR).
  • the pharmaceutical composition comprising the circular polyribonucleotide or modified RNA is at least 30% (w/w), 40% (w/w), 50% (w/w), 60% (w/w), 70% (w/w), 80% (w/w), 85% (w/w), 90% (w/w), 91% (w/w), 92% (w/w), 93% (w/w), 94% (w/w), 95% (w/w), 96% (w/w), 97% (w/w), 98% (w/w), 99% (w/w), or 100% (w/w) pure on a mass basis.
  • a concentration of the circular polyribonucleotide or modified RNA in the pharmaceutical composition is at least 0.1 ng/mL, 0.5 ng/mL, 1 ng/mL, 5 ng/mL, 10 ng/mL, 50 ng/mL, 0.1 ⁇ g/mL, 0.5 ⁇ g/mL, 1 ⁇ g/mL, 2 ⁇ g/mL, 5 ⁇ g/mL, 10 ⁇ g/mL, 20 ⁇ g/mL, 30 ⁇ g/mL, 40 ⁇ g/mL, 50 ⁇ g/mL, 60 ⁇ g/mL, 70 ⁇ g/mL, 80 ⁇ g/mL, 100 ⁇ g/mL, 200 ⁇ g/mL, 300 ⁇ g/mL, 500 ⁇ g/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, 5 mg/mL, 10 mg/mL, 100 mg/mL, or 500 mg/mL.
  • the pharmaceutical composition is substantially free of mononucleotide or has a mononucleotide content of no more than 1 pg/ml, 10 pg/ml, 0.1 ng/ml, 1 ng/ml, 5 ng/ml, 10 ng/ml, 15 ng/ml, 20 ng/ml, 25 ng/ml, 30 ng/ml, 35 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 1000 ⁇ g/mL, 5000 ⁇ g/mL, 10,000 ⁇ g/mL, or 100,000 ⁇ g/mL.
  • the pharmaceutical composition has a mononucleotide content of no more than 0.1% (w/w), 0.2% (w/w), 0.3% (w/w), 0.4% (w/w), 0.5% (w/w), 0.6% (w/w), 0.7% (w/w), 0.8% (w/w), 0.9% (w/w), 1% (w/w), 2% (w/w), 3% (w/w), 4% (w/w), 5% (w/w), 6% (w/w), 7% (w/w), 8% (w/w), 9% (w/w), 10% (w/w), 15% (w/w), 20% (w/w), 25% (w/w), 30% (w/w), or any percentage therebetween of total nucleotides on a mass basis, wherein total nucleotide content is the total mass of deoxyribonucleotide molecules and ribonucleotide molecules.
  • the pharmaceutical composition has a linear RNA content, e.g., linear RNA counterpart or RNA fragments, of no more than 1 ng/ml, 5 ng/ml, 10 ng/ml, 15 ng/ml, 20 ng/ml, 25 ng/ml, 30 ng/ml, 35 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 1 ⁇ g/ml, 10 ⁇ g/ml, 50 ⁇ g/ml, 100 ⁇ g/ml, 200 g/ml, 300 ⁇ g/ml, 400 ⁇ g/ml, 500 ⁇ g/ml, 600 ⁇ g/ml, 700 ⁇ g/ml,
  • the pharmaceutical composition has a nicked RNA content of no more than 10% (w/w), 9.9% (w/w), 9.8% (w/w), 9.7% (w/w), 9.6% (w/w), 9.5% (w/w), 9.4% (w/w), 9.3% (w/w), 9.2% (w/w), 9.1% (w/w), 9% (w/w), 8% (w/w), 7% (w/w), 6% (w/w), 5% (w/w), 4% (w/w), 3% (w/w), 2% (w/w), 1% (w/w), 0.5% (w/w), or 0.1% (w/w), or percentage therebetween.
  • the pharmaceutical composition is substantially free of DNA content, e.g., template DNA or cell DNA (e.g., host cell DNA), has a DNA content, as low as zero, or has a DNA content of no more than 1 pg/ml, 10 pg/ml, 0.1 ng/ml, 1 ng/ml, 5 ng/ml, 10 ng/ml, 15 ng/ml, 20 ng/ml, 25 ng/ml, 30 ng/ml, 35 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 1000 ⁇ g/mL, 5000 ⁇ g/mL, 10,000 ⁇ g/mL, or 100,000 ⁇ g/mL.
  • DNA content e.g., template
  • the pharmaceutical composition is substantially free of an impurity (e.g., a cell protein, a cell nucleic acid, an enzyme, a reagent component, a gel component, or a chromatographic material, protein contamination, or endotoxin contamination).
  • the pharmaceutical composition comprises a concentration of protein that is less than 0.1 ng, 1 ng, 5 ng, 10 ng, 15 ng, 20 ng, 25 ng, 30 ng, 35 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 200 ng, 300 ng, 400 ng, or 500 ng of protein per milligram (mg) of the circular polyribonucleotide.
  • an impurity e.g., a cell protein, a cell nucleic acid, an enzyme, a reagent component, a gel component, or a chromatographic material, protein contamination, or endotoxin contamination.
  • the pharmaceutical composition comprises a concentration of protein that is
  • the pharmaceutical composition has an A260/A280 absorbance ratio from about 1.6 to about 2.3, e.g., as measured by spectrophotometer. In some embodiments, the A260/A280 absorbance ratio is about 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, or any number therebetween. In some embodiments, the pharmaceutical composition comprises an amount of endotoxin that is less than 20 EU/kg (weight), 10 EU/kg, 5 EU/kg, 1 EU/kg, or is below a predetermined threshold, e.g., the pharmaceutical composition comprises a level of endotoxin below a limit of detection by a specified method.
  • the pharmaceutical composition is a sterile drug product or substantially free of microorganisms (e.g., supports growth of fewer than 100 viable microorganisms as tested under aseptic conditions).
  • the pharmaceutical composition comprises a bioburden of less than 100 CFU/100 mL, 50 CFU/100 mL, 40 CFU/100 mL, 30 CFU/100 mL, 200 CFU/100 mL, 10 CFU/100 mL, or 10 CFU/100 mL before sterilization.
  • the pharmaceutical composition comprising the circular polyribonucleotide that has undergone a purification step produces a reduced level of one more markers of an immune or inflammatory response after administration to a subject compared to a pharmaceutical composition comprising an unpurified circular polyribonucleotide.
  • the one or more markers of an immune or inflammatory response is a cytokine or immune response related gene.
  • the one or more markers of an immune or inflammatory response is expression of a gene, such as RIG-I, MDA5, PKR, IFN-beta, OAS, and OASL. III.
  • Circular Polyribonucleotides and Modified RNAs Also provided herein are methods for treating or reducing the risk of developing a GLP- 2-related disease in a subject that include administering to the subject a therapeutically effective amount of any of the circular polyribonucleotides or modified RNAs described herein, or any of the pharmaceutical compositions described herein.
  • GLP-2 related disease refers to any disease in which cells engage in aberrant GLP-2 signaling, e.g., aberrant insulin signaling.
  • Non-limiting examples of GLP-2-related diseases include short bowel syndrome (SBS), short bowel syndrome with intestinal failure (SBS-IF), pediatric SBS, adult SBS, major small bowel resection, total parenteral nutrition (TPN)-induced intestinal hypoplasia, small and large intestinal inflammation, Crohn’s disease, chemotherapy-induced mucositis, and intestinal ischemia–reperfusion injury.
  • SBS short bowel syndrome
  • SBS-IF short bowel syndrome with intestinal failure
  • TPN total parenteral nutrition
  • an effective amount of any of the circular polyribonucleotides, modified RNAs, or pharmaceutical compositions described herein is administered to a subject (e.g., a mammalian subject, e.g., a human) having or at risk for developing a GLP-2 related disease (e.g., short bowel syndrome) via a suitable route (e.g., local administration and/or systemic administration).
  • a subject e.g., a mammalian subject, e.g., a human
  • a suitable route e.g., local administration and/or systemic administration.
  • the subject has been previously identified or diagnosed as having a GLP-2- related disease.
  • the subject has been previously identified as having an elevated risk of developing a GLP-2-related disease. LRN Ref.
  • subject refers to a subject who needs treatment as described herein.
  • the subject is a human or a non-human mammal (e.g., cat, dog, horse, cow, goat, or sheep).
  • a human subject who needs treatment can be a human patient having, suspected of having, or at risk for having a GLP-2-related disease, e.g., short bowel syndrome (SBS), short bowel syndrome with intestinal failure (SBS-IF), pediatric SBS, adult SBS, major small bowel resection, total parenteral nutrition (TPN)-induced intestinal hypoplasia, small and large intestinal inflammation, Crohn’s disease, chemotherapy-induced mucositis, and intestinal ischemia–reperfusion injury
  • An “effective amount” or a “therapeutically effective amount” as used herein refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents.
  • Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known in the medical field. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons, or virtually any other reason. Empirical considerations such as the half-life of an agent will generally contribute to the determination of the dosage.
  • Frequency of administration can be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or amelioration and/or delay of a GLP-2-related disease (e.g., short bowel syndrome (SBS), short bowel syndrome with intestinal failure (SBS-IF), pediatric SBS, adult SBS, major small bowel resection, total parenteral nutrition (TPN)-induced intestinal hypoplasia, small and large intestinal inflammation, Crohn’s disease, chemotherapy-induced mucositis, intestinal ischemia–reperfusion injury, or a combination thereof).
  • a GLP-2-related disease e.g., short bowel syndrome (SBS), short bowel syndrome with intestinal failure (SBS-IF), pediatric SBS, adult SBS, major small bowel resection, total parenteral nutrition (TPN)-induced intestinal hypoplasia, small and large intestinal inflammation, Crohn’s disease, chemotherapy-induced mucositis, intestinal ischemia–reperfusion injury, or a combination thereof).
  • dosages of any of the circular polyribonucleotides, modified RNAs, or pharmaceutical compositions described herein can be determined empirically in individuals who have been given one or more administration(s) of the circular polyribonucleotide LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 or modified RNA.
  • individuals are given incremental dosages of any of the circular polyribonucleotides, modified RNAs, and pharmaceutical compositions described herein and an indicator and/or a symptom of a GLP-2-related disease can be followed to assess efficacy.
  • Any suitable dosing regimen can be used in methods described herein.
  • the dosage regimen depends on the pattern of pharmacokinetic decay that the practitioner wishes to achieve.
  • dosing frequency is once every day, once every other day, once every week, or longer. In some embodiments, dosing frequency is multiple times per day.
  • the treatment can be sustained until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved to alleviate a GLP-2-related disease, or a symptom thereof.
  • dosing regimens (including inhibitor used) can vary over time.
  • a circular or linear polyribonucleotide described herein may be administered in a single dose or in multiple doses to a cell, tissue or subject.
  • a circular or linear polydeoxyribonucleotide described herein may be administered in a single dose or in multiple doses to a cell, tissue or subject.
  • a method of administering multiple doses of a composition of a nucleic acid molecule described herein e.g., a circular polyribonucleotide, a linear polyribonucleotide, a circular polydeoxyribonucleotide, a linear polydeoxyribonucleotide
  • a pharmaceutical composition comprises providing two or more compositions, over a period of time, to a cell, tissue or subject (e.g. a mammal).
  • multiple doses of a composition of a nucleic acid molecule described herein may be administered to a subject over a defined time course.
  • a composition of a nucleic acid molecule described herein e.g., a circular polyribonucleotide, a linear polyribonucleotide, a circular polydeoxyribonucleotide, a linear polydeoxyribonucleotide
  • a pharmaceutical composition e.g., in a pharmaceutical composition
  • the methods according to this aspect of the invention comprise sequentially administering to a subject multiple doses of a composition of a nucleic acid molecule described herein (e.g., a circular polyribonucleotide, a linear polyribonucleotide, a circular polydeoxyribonucleotide, a linear polydeoxyribonucleotide) (e.g., in a pharmaceutical composition).
  • a composition of a nucleic acid molecule described herein e.g., a circular polyribonucleotide, a linear polyribonucleotide, a circular polydeoxyribonucleotide, a linear polydeoxyribonucleotide
  • compositions of a nucleic acid molecule described herein e.g., a circular polyribonucleotide, a linear polyribonucleotide, a circular polydeoxyribonucleotide, a linear polydeoxyribonucleotide
  • a pharmaceutical composition e.g., in a pharmaceutical composition
  • LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 e.g., hours, days, weeks or months.
  • the present invention provides methods which comprise sequentially administering to the subject a single initial dose of a composition of a nucleic acid molecule described herein (e.g., a circular polyribonucleotide, a linear polyribonucleotide, a circular polydeoxyribonucleotide, a linear polydeoxyribonucleotide) (e.g., in a pharmaceutical composition), followed by one or more secondary doses of the composition, and optionally followed by one or more tertiary doses of the composition.
  • a composition of a nucleic acid molecule described herein e.g., a circular polyribonucleotide, a linear polyribonucleotide, a circular polydeoxyribonucleotide, a linear polydeoxyribonucleotide
  • initial dose refers to the temporal sequence of administration of a composition of a nucleic acid molecule described herein (e.g., a circular polyribonucleotide, a linear polyribonucleotide, a circular polydeoxyribonucleotide, a linear polydeoxyribonucleotide) (e.g., in a pharmaceutical composition).
  • a composition of a nucleic acid molecule described herein e.g., a circular polyribonucleotide, a linear polyribonucleotide, a circular polydeoxyribonucleotide, a linear polydeoxyribonucleotide
  • the “initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”); the “secondary doses” are the doses which are administered after the initial dose; and the “tertiary doses” are the doses which are administered after the secondary doses.
  • the initial, secondary, and tertiary doses may all contain the same amount of a composition of a nucleic acid molecule described herein (e.g., a circular polyribonucleotide, a linear polyribonucleotide, a circular polydeoxyribonucleotide, a linear polydeoxyribonucleotide) (e.g., in a pharmaceutical composition), and in certain embodiments, may differ from one another in terms of frequency of administration.
  • a composition of a nucleic acid molecule described herein e.g., a circular polyribonucleotide, a linear polyribonucleotide, a circular polydeoxyribonucleotide, a linear polydeoxyribonucleotide
  • the amount of a composition of a nucleic acid molecule described herein e.g., a circular polyribonucleotide, a linear polyribonucleotide, a circular polydeoxyribonucleotide, a linear polydeoxyribonucleotide
  • a pharmaceutical composition e.g., in a pharmaceutical composition
  • one or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as “loading doses” followed by subsequent doses that are administered on a less frequent basis (e.g., “maintenance doses”).
  • each secondary and/or tertiary dose is administered after the immediately preceding dose.
  • the immediately preceding dose means, in a sequence of multiple administrations, the dose of the composition of a nucleic acid molecule described herein (e.g., a circular polyribonucleotide, a linear polyribonucleotide, a circular polydeoxyribonucleotide, a linear polydeoxyribonucleotide) (e.g., in a pharmaceutical LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 composition) which is administered to a subject prior to the administration of the very next dose in the sequence with no intervening doses.
  • a nucleic acid molecule described herein e.g., a circular polyribonucleotide, a linear polyribonucleotide, a circular polydeoxyribonucleotide, a linear polydeoxyribonucleotide
  • each secondary and/or tertiary dose is administered every day, every 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the immediately preceding dose. In certain embodiments, each secondary and/or tertiary dose is administered every 0.5 weeks, 1 week, 2 weeks, 3 weeks, or 4 weeks after the immediately preceding dose.
  • the methods according to this aspect of the invention may comprise administering to a subject any number of secondary and/or tertiary doses of a composition of a nucleic acid molecule described herein (e.g., a circular polyribonucleotide, a linear polyribonucleotide, a circular polydeoxyribonucleotide, a linear polydeoxyribonucleotide) (e.g., in a pharmaceutical composition).
  • a composition of a nucleic acid molecule described herein e.g., a circular polyribonucleotide, a linear polyribonucleotide, a circular polydeoxyribonucleotide, a linear polydeoxyribonucleotide
  • only a single secondary dose is administered to the subject.
  • two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the subject.
  • tertiary dose only a single tertiary dose is administered to the subject.
  • two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the subject.
  • the frequency at which the secondary and/or tertiary doses are administered to a subject can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment.
  • multiple doses are provided to produce a level of the composition or express a level of the GLP-2 or GLP-2 analog in a cell, tissue or subject.
  • multiple doses are provided to produce or maintain a level of the composition, or to produce or maintain a level of the GLP-2 or GLP-2 analog, in a cell, tissue or subject for a period of time, for instance, for at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 21, or 24 months, or at least 1, 2, 3, 4, or 5 years.
  • the method comprises providing (e.g., administering) at least a first composition and a second composition to the cells, tissue, or subject (e.g., a mammal, e.g., a human).
  • the method further comprises providing (e.g., administering) a third composition, fourth composition, fifth composition, sixth composition, seventh composition, eighth composition, ninth composition, tenth composition, or more.
  • additional compositions are provided for the duration of the life of the cell.
  • additional compositions are provided (e.g., administered) while the cell, LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 tissue or subject obtains a benefit from the composition, e.g., stimulated insulin secretion, altered metabolism, improved glucose homeostasis, reduced body weight, improved liver function, and/or amelioration of fibrosis.
  • a first composition in a multiple dosing regimen comprises a first amount of the nucleic acid molecule (e.g., circular polyribonucleotide) disclosed herein.
  • a second composition in a multiple dosing regimen comprises a second amount of the nucleic acid molecule (e.g., circular polyribonucleotide) disclosed herein.
  • a third composition, a fourth composition, a fifth composition, a sixth composition, a seventh composition, an eighth composition, a ninth composition, a tenth composition, or more in a multiple dosing regimen comprises a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more amount of the nucleic acid molecule (e.g., circular polyribonucleotide) disclosed herein.
  • the second amount of the nucleic acid molecule e.g., circular polyribonucleotide
  • the second amount of the nucleic acid molecule is the same as the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide).
  • the third amount of the nucleic acid molecule is the same as the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide).
  • the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amount of the nucleic acid molecule is the same as the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide).
  • the second amount of the nucleic acid molecule is less than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide).
  • the third amount of the nucleic acid molecule is less than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide).
  • the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amount of the nucleic acid molecule is less than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide).
  • the second amount of the nucleic acid molecule is greater than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide).
  • the third amount of the nucleic acid molecule is greater than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide).
  • the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amount of the nucleic acid molecule is greater than the first amount of the nucleic acid molecule (e.g., circular polyribonucleotide).
  • an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of the second composition varies by no more than 1%, 5%, 10%, 15%, 20%, or 25% of an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of the first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of the second composition is no more than 1%, 5%, 10%, 15%, 20%, or 25% less than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of the first composition.
  • an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a second composition is from 0.1-fold to 1000-fold higher than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition. In some embodiments, an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a second composition is 0.1-fold, 1-fold, 5-fold, 10-fold, 100-fold, or 1000-fold higher than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition.
  • an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a subsequent composition is 0.1-fold, 1-fold, 5-fold, 10-fold, 100-fold, or 1000-fold higher than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition.
  • an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a second composition is from 0.1-fold to 1000-fold lower than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition.
  • an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a second composition is 0.1-fold, 1-fold, 5-fold, 10-fold, 100-fold, or 1000-fold lower than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition.
  • an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a subsequent composition is 0.1-fold, 1-fold, 5-fold, 10-fold, 100-fold, or 1000-fold lower than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition.
  • an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a subsequent composition is from 0.1-fold to 1000-fold higher or lower than an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition.
  • an amount of the nucleic acid molecule e.g., circular LRN Ref. No.: LRN23-107WO Fish Ref.
  • No.: 56929-0005WO1 polyribonucleotide) of a subsequent composition e.g., after a first composition of an amount of nucleic acid molecule (e.g., circular polyribonucleotide)
  • a subsequent composition e.g., after a first composition of an amount of nucleic acid molecule (e.g., circular polyribonucleotide)
  • an amount of nucleic acid molecule e.g., circular polyribonucleotide
  • a first composition comprises 1-fold nucleic acid molecule (e.g., circular polyribonucleotide), a second composition comprises 5-fold nucleic acid molecule (e.g., circular polyribonucleotide) compared to the first composition, and a third composition comprises 0.2-fold nucleic acid molecule (e.g., circular polyribonucleotide) compared to the first composition.
  • the second composition comprises at least 5-fold nucleic acid molecule (e.g., circular polyribonucleotide) compared to an amount of nucleic acid molecule (e.g., circular polyribonucleotide) of a first composition.
  • the first composition comprises a higher amount of the nucleic acid molecule (e.g., circular polyribonucleotide) than the second composition. In some embodiments, the first composition comprises a higher amount of the nucleic acid molecules (e.g., circular polyribonucleotides) than the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth composition. In some embodiments, the plurality (e.g., two or more) of compositions of a nucleic acid molecule (e.g., circular polyribonucleotide) encoding a GLP-2 or GLP-2 analog, which are administered in a multiple dosing regimen as described herein, are the same compositions.
  • the nucleic acid molecule e.g., circular polyribonucleotide
  • the plurality (e.g., two or more) of compositions of a nucleic acid molecule (e.g., circular polyribonucleotide) encoding a GLP-2 or GLP-2 analog, which are administered in a multiple dosing regimen as described herein, are different compositions.
  • the same compositions comprise the nucleic acid molecules (e.g., circular polyribonucleotides) encoding the same GLP-2 or GLP-2 analog.
  • the different compositions comprise the nucleic acid molecules (e.g., circular polyribonucleotides) encoding different a GLP-2 or GLP-2 analog, or a combination thereof.
  • a composition of the nucleic acid molecule (e.g., a circular polyribonucleotide) disclosed herein can induce a response in a subject.
  • the response may be stimulated insulin secretion, altered metabolism, improved glucose homeostasis, reduced body weight, improved liver function, and/or amelioration of fibrosis.
  • the method of administering the nucleic acid molecule (e.g., circular polyribonucleotide) provided herein includes administering LRN Ref. No.: LRN23-107WO Fish Ref.
  • the method provided herein includes administering to a subject in need thereof the nucleic acid molecule for at least 3 times, with an interval of about 7 days.
  • a level of the GLP-2 or GLP-2 analog e.g., a plasma GLP-2 or GLP-2 level
  • a level with variation of less than 50%, 40%, 30%, 20%, or 10% is maintained at a level with variation of less than 50%, 40%, 30%, 20%, or 10% for a period of longer than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, or 20 weeks after the last dose.
  • a level of the GLP-2 or GLP-2 analog e.g., a plasma GLP-2 or GLP-2 level
  • a first level is maintained at a first level for a period of longer than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, or 20 weeks after the second, third, fourth, fifth, sixth, seventh, eight, or the last dose, wherein the first level is higher than a level of the GLP-2 or GLP-2 analog measured shortly after the first dose (e.g., measured about 12, 24, 36, or 48 hours after the first dose).
  • a level of the GLP-2 or GLP-2 analog e.g., a plasma GLP-2 or GLP-2 level
  • a level of the GLP-2 or GLP-2 analog is maintained at a first level for a period of longer than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks after the second, third, fourth, fifth, sixth, seventh, eight, or the last dose, wherein the first level is higher than a level of the GLP-2 or GLP-2 analog measured shortly after the first dose (e.g., measured about 12, 24, 36, or 48 hours after the first dose).
  • the term “treating” refers to the application or administration of a composition including one or more active agents to a subject who has a GLP-2-related disease (e.g., short bowel syndrome (SBS), short bowel syndrome with intestinal failure (SBS-IF), pediatric SBS, adult SBS, major small bowel resection, total parenteral nutrition (TPN)-induced intestinal hypoplasia, small and large intestinal inflammation, Crohn’s disease, chemotherapy- induced mucositis, intestinal ischemia–reperfusion injury, or a combination thereof), a symptom of a GLP-2-related disease, and/or an increased risk of developing a GLP-2-related disease, with LRN Ref. No.: LRN23-107WO Fish Ref.
  • a GLP-2-related disease e.g., short bowel syndrome (SBS), short bowel syndrome with intestinal failure (SBS-IF), pediatric SBS, adult SBS, major small bowel resection, total parenteral nutrition (TPN)-induced intestinal hypoplasia
  • No.: 56929-0005WO1 the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the GLP-2-related disease, the symptom of the GLP-2 related disease, and/or the risk of developing a GLP-2-related disease.
  • Alleviating a GLP-2-related disease includes delaying the development or progression of the disease, and/or reducing disease severity. Alleviating the disease does not necessarily require curative results.
  • “delaying” the development of a GLP-2-related disease e.g., short bowel syndrome (SBS), short bowel syndrome with intestinal failure (SBS-IF), pediatric SBS, adult SBS, major small bowel resection, total parenteral nutrition (TPN)-induced intestinal hypoplasia, small and large intestinal inflammation, Crohn’s disease, chemotherapy-induced mucositis, intestinal ischemia–reperfusion injury, or a combination thereof
  • SBS short bowel syndrome
  • SBS-IF short bowel syndrome with intestinal failure
  • TPN total parenteral nutrition
  • This delay can be of varying lengths of time, depending on the history of the GLP-2-related disease and/or individuals being treated.
  • a method that “delays” or alleviates the development of a GLP-2-related disease and/or delays the onset of the GLP-2-related disease is a method that reduces probability of developing one or more symptoms of the GLP-2-related disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result. “Development” or “progression” of a disease means initial manifestations and/or ensuing progression of the GLP-2-related disease. Development of the GLP-2-related disease can be detectable and assessed using standard clinical techniques known in the art. However, development also refers to progression that may be undetectable.
  • any of the circular polyribonucleotides, modified RNAs, or pharmaceutical compositions described herein is administered to a subject in an amount sufficient to increase levels of GLP-2-mediated signaling by at least 5% (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more).
  • LRN Ref LRN Ref.
  • any of the circular polyribonucleotides, modified RNAs, or pharmaceutical compositions described herein is administered to a subject in an amount sufficient to increase intestinal growth in a subject by at least 5% (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more).
  • increasing intestinal growth includes increasing intestinal tissue and/or cells.
  • the cells are crypt cells.
  • any of the circular polyribonucleotides, modified RNAs, or pharmaceutical compositions described herein is administered to a subject in an amount sufficient to increase intestinal absorption of fluids and nutrients in a subject by at least 5% (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more).
  • methods for treating a GLP-2-related disease involve restoring, at least in part, expression and/or activity of GLP-2 using any of the circular polyribonucleotides, modified RNAs, or pharmaceutical compositions described herein.
  • any of the circular polyribonucleotides, modified RNAs, or pharmaceutical compositions described herein can be administered using any suitable method for achieving delivery of the circular polyribonucleotide or modified RNA to the subject in need thereof.
  • the route of administration can depend on various factors such as the type of GLP-2-related disease to be treated and the site of the disease.
  • any of the circular polyribonucleotides, modified RNAs, or pharmaceutical composition described herein can be administered topically, locally, nasally, parenterally, buccally, or by inhalation.
  • Parenteral administration includes, but is not limited to, subcutaneous, intracutaneous, intravenous, intramuscular, or intrasynovial injection, or infusion techniques.
  • any of the circular polyribonucleotides, modified RNAs, or pharmaceutical composition described herein is administered locally (e.g., intramuscular injection). In some embodiments, any of the circular polyribonucleotides, the modified RNAs, or the pharmaceutical composition described herein is administered systemically (e.g., intravenous infusion). In some embodiments, any of the circular polyribonucleotides, the modified RNAs, or the pharmaceutical compositions described herein is administered one or more times to the subject. Alternatively, or in addition, any of the circular polyribonucleotides, the modified RNAs, or the LRN Ref. No.: LRN23-107WO Fish Ref.
  • kits described herein can be administered as part of a combination therapy with an additional therapeutic agent.
  • Any therapeutic agent suitable for treating a GLP-2-related disease can be used as an additional therapeutic agent in methods and/or pharmaceutical compositions described herein.
  • additional therapeutic agents include anti-inflammatory agents (e.g., steroids, such as corticosteroids)), immunosuppressants (e.g., methotrexate, cyclosporine), or insulin.
  • anti-inflammatory agents e.g., steroids, such as corticosteroids
  • immunosuppressants e.g., methotrexate, cyclosporine
  • insulin e.g., insulin
  • no other agents are administered.
  • kits that can be used, e.g., in any of the methods described herein.
  • kits include (a) (i) a composition comprising any of the circular polyribonucleotides described herein or any of the modified RNAs described herein or (ii) any of the pharmaceutical compositions described herein; and optionally, (b) instructions for administering the composition or the pharmaceutical composition to a subject in need thereof.
  • Nucleic Acids The present disclosure further provides nucleic acids (e.g., DNA or RNA) encoding any of the polypeptides described herein.
  • the nucleic acid encodes a polypeptide comprising (i) a glucagon-like peptide-2 (GLP-2) polypeptide or analog thereof (e.g., any of the GLP-2 polypeptides or analogs described herein), (ii) a half-life extension moiety (e.g., any of the exemplary half-life extension moieties described herein), and (iii) a secretion signal sequence (e.g., any of the exemplary secretion signal sequences described herein).
  • GLP-2 glucagon-like peptide-2
  • a half-life extension moiety e.g., any of the exemplary half-life extension moieties described herein
  • a secretion signal sequence e.g., any of the exemplary secretion signal sequences described herein.
  • the nucleic acid encodes a polypeptide comprising (i) a glucagon-like peptide-2 (GLP-2) polypeptide or analog thereof (e.g., any of the exemplary GLP- 2 polypeptides or analogs described herein) and (ii) a half-life extension moiety (e.g., any of the exemplary half-life extension moieties described herein).
  • the nucleic acid encodes a polypeptide comprising a glucagon-like peptide-2 (GLP-2) polypeptide or analog thereof (e.g., any of exemplary the GLP-2 polypeptides or analogs described herein).
  • the present disclosure also provides polypeptides generated from any of the circular polyribonucleotides, any of the modified RNAs, and any of the nucleic acids described herein.
  • the polypeptide comprises (i) a glucagon-like peptide-2 (GLP-2) polypeptide or analog thereof (e.g., any of the exemplary GLP-2 polypeptides or analogs described herein), (ii) a half-life extension moiety (e.g., any of the exemplary half-life extension moieties described herein), and (iii) a secretion signal sequence (e.g., any of the exemplary secretion signal sequences described herein).
  • GLP-2 glucagon-like peptide-2
  • a half-life extension moiety e.g., any of the exemplary half-life extension moieties described herein
  • a secretion signal sequence e.g., any of the exemplary secretion signal sequences described herein.
  • the polypeptide comprises (i) a glucagon-like peptide-2 (GLP-2) polypeptide or analog thereof (e.g., any of the exemplary GLP-2 polypeptides or analogs described herein) and (ii) a half-life extension moiety (e.g., any of the exemplary half-life extension moieties described herein).
  • the polypeptide comprises a glucagon-like peptide-2 (GLP-2) polypeptide or analog thereof (e.g., any of the exemplary GLP-2 polypeptides or analogs described herein).
  • the polypeptides can include an IgG constant domain (e.g., IgG1, IgG2, IgG3, IgG4), a serum protein or fragment thereof (e.g., albumin or fragment thereof (e.g., human serum albumin or a fragment thereof), fibronectin or fragment thereof (e.g., human fibronectin or a fragment thereof), or transferrin or fragment thereof (e.g., human transferrin or a fragment thereof)), and an antibody or an antigen-binding fragment thereof that binds to a serum protein (e.g., an antibody or antigen-binding fragment thereof that binds to human albumin, human fibronectin, or human transferrin).
  • IgG constant domain e.g., IgG1, IgG2, IgG3, IgG4
  • a serum protein or fragment thereof e.g., albumin or fragment thereof (e.g., human serum albumin or a fragment thereof), fibronectin or fragment thereof (e.g.,
  • the IgG constant domain includes a half-life extension substitution (e.g., any of the half-life extension substitutions described herein).
  • a half-life extension substitution e.g., any of the half-life extension substitutions described herein.
  • GGGGS GGGGS3 (SEQ ID NO: 141) linker (also referred to as teduglutide-Fc). Recombinant teduglutide was used as a control.
  • the circular RNA construct was transfected into HeLa cells using LipofectamineTM MessengerMAXTM (Invitrogen LMRNA001) according to the manufacturer’s instructions. Activity was measured using a G protein-coupled receptor (GPCR) assay in which cyclic adenosine monophosphate (cAMP) production were measured with a fluorescently labelled cAMP probe.
  • GPCR G protein-coupled receptor
  • teduglutide-Fc expressed from circular RNA showed approximately 3-fold reduced activity level as compared to recombinant teduglutide in a GPCR assay.
  • a functionally active GLP-2 analog can be expressed from circular RNA.
  • Example 2 Expression of Functionally Active GLP-2 Analog from Circular RNA in Mammalian Cells The expression of the GLP-2 analog teduglutide from various circular RNA constructs (Table 8) was tested in mammalian cells (HEK293T cells). Circular RNA constructs were transfected into HeLa cells using LipofectamineTM MessengerMAXTM (Invitrogen LMRNA001) according to the manufacturer’s instructions.
  • Table 9 Circular RNA constructs Construct Polypeptide IRES C-Terminal CE 5’ Spacer 3’ Spacer K274(Shire Modified ne poly(adenosine-uridine) spacer sequence having a length of 120 nucleotides; CE: circularization element; Anabaena2.3: A specific intron based circularization element; Anabaena2: A specific intron based circularization element.
  • LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 Taken together, these results demonstrate that functionally active teduglutide can be expressed from various circular RNA constructs.
  • Example 3 Pharmacokinetic (PK) Profiles of GLP-2 Fc Expressed from Circular RNA Compared to Recombinant GLP-2 Analogs
  • the PK profile of teduglutide-Fc expressed from circular RNA encoding teduglutide-Fc was evaluated in mice. Circular RNA encoding teduglutide-Fc (1.5 mg/kg) was injected into mice via subcutaneous injection in the interscapular area. Blood was collected from the tail-vein of each mouse. Serum was collected from blood and the concentration and pharmacokinetic (PK) parameters for teduglutide-Fc were determined.
  • PK Pharmacokinetic
  • PK profile of teduglutide-Fc expressed from circular RNA in mice was then compared to the reported PK profiles of various recombinant GLP-2 analogs.
  • Reported PK profiles were obtained from Hargrove et al., “Pharmacological Characterization of Apraglutide, a Novel Long-Acting Peptidic Glucagon-Like Peptide-2 Agonist, for the Treatment of Short Bowel Syndrome,” J Pharmacol Exp Ther;373(2):193-203 (2020) and Choi et al., “HM15912, a Novel Long-acting Glucagon-like Peptide-2 Analog, Improves Intestinal Growth and Absorption Capacity in a Male Rat Model of Short Bowel Syndrome,” J Pharmacol Exp Ther; JPET-AR- 2022-001381 (2022).
  • Example 4 Characterization of Lipid Nanoparticle (LNP) Formulations Various LNP formulations were tested for tolerability and expression of teduglutide-Fc. Circular RNA encoding teduglutide-Fc (4133_3) was formulated in different LNPs. LNP formulations (0.75 mg/kg) were injected into mice. Negative control mice were injected with PBS. Blood was collected from the tail-vein at 4 hours, 6 hours, and 24 hours after injection. Expression of teduglutide-Fc at 6 and 24 hours (FIG. 4A), complete blood cell count (CBC) at 24 hours (FIGs. 4B-4C), ALT levels at 24 hours (FIG. 4D), IFN ⁇ levels at 4 hours (FIG.
  • Example 5 Dose Response in Mice Circular RNA (7297) and mRNA containing the modified nucleoside N1- methylpseudoiridine (m1 ⁇ ) (7377) encoding teduglutide-Fc was formulated into LNPs (LRN28). A dose response of the LNP formulations of teduglutide-Fc was evaluated in either WT or NSG (NOD-scid IL2Rg null ) mice.
  • Circular RNA in LNPs encoding teduglutide-Fc (1, 0.3, 0.1, or 0.03 mg/kg) in WT and NSG mice and m1 ⁇ mRNA in LNP encoding teduglutide-Fc (0.3 mg/kg) in NSG mice only were injected via subcutaneous injection in the interscapular area. Blood was collected from the tail-vein of each mouse at baseline and 6h, D1, D2, D10, D17, and D28 after subcutaneous administration.
  • Expression of GLP2-Fc (FIGs. 5A-B), cytokine levels (FIGs. 5C- F), and anti-target antibody(ATA) response (FIG. 5G) were measured at various timepoints.
  • Example 6 Characterization of GLP-2 Fc Expressed from Circular RNA in Non-Human Primates Circular RNA (7297) and mRNA containing the modified nucleoside N1- methylpseudoiridine (m1 ⁇ ) (7377) encoding teduglutide-Fc was formulated into LNPs (LRN28).
  • the circular RNA formulations had 2 purification levels: pre-Reverse Phase (pre-RP) or reverse phase (RP).
  • pre-RP pre-Reverse Phase
  • RP reverse phase
  • the LNP formulations of teduglutide-Fc were intravenously dosed in non-human primates (NHPs) at 0.5 mg/kg with or without steroids, as shown in Table 11.
  • the steroid if given, was dexamethasone intramuscularly administered at 1mpk 24h and 1h pre-dose and 24h LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 post-dose.
  • Blood was collected at baseline (D -14), post-infusion, 6hr, 24h, 48h, 72h, D5, D7, D14, D21, and D28.
  • Teduglutide-Fc expression FIG. 6A
  • antibody production FIG. 6B
  • complete blood count including lymphocyte count (FIG. 6C), cytokine responses (FIG. 6D), CRP levels (FIG. 6E), and activate partial thromboplastin (FIG. 6F) were measured.
  • Embodiment 1 is a circular polyribonucleotide comprising a sequence encoding a polypeptide comprising (i) a glucagon-like peptide-2 (GLP-2) polypeptide or analog thereof, (ii) a half-life extension moiety, and (iii) a secretion signal sequence.
  • GLP-2 glucagon-like peptide-2
  • Embodiment 2 is the circular polyribonucleotide of embodiment 1, wherein the GLP-2 polypeptide or analog thereof comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.
  • Embodiment 3 is the circular polyribonucleotide of embodiment 1 or 2, wherein the GLP- 2 polypeptide or analog thereof comprises an amino acid sequence of any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.
  • Embodiment 4 is the circular polyribonucleotide of any one of embodiments 1-3, wherein the sequence encoding the polypeptide comprises a nucleic acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.
  • Embodiment 5 is the circular polyribonucleotide of any one of embodiments 1-4, wherein the sequence encoding the polypeptide comprises a nucleic acid sequence of any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.
  • Embodiment 6 is the circular polyribonucleotide of any one of embodiments 1-5, wherein the GLP-2 analog comprises teduglutide, glepaglutide, dapiglutide, apraglutide, HM15912, and SHP-681.
  • Embodiment 7 is the circular polyribonucleotide of any one of embodiments 1-6, wherein the half-life extension moiety comprises a peptide moiety.
  • Embodiment 8 is the circular polyribonucleotide of embodiment 7, wherein the peptide moiety comprises an Fc domain, an albumin polypeptide, a nanobody, or a post-translational modification site.
  • Embodiment 9 is the circular polyribonucleotide of embodiment 8, wherein the Fc domain comprises an IgG1 Fc domain, an IgG2 Fc domain, an IgG3 Fc domain, or an IgG4 Fc domain.
  • Embodiment 10 is the circular polyribonucleotide of embodiment 8 or 9, wherein the Fc domain comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identical to SEQ ID NO: 15 (IgG1 Fc domain), SEQ ID NO: 17 (IgG2 Fc domain), SEQ ID NO: 19 (IgG3 Fc domain), or SEQ ID NO: 21 (IgG4 Fc domain).
  • Embodiment 11 is the circular polyribonucleotide of any one of embodiments 8-10, wherein the Fc domain comprises an amino acid sequence of SEQ ID NO: 15 (IgG1 Fc domain), SEQ ID NO: 17 (IgG2 Fc domain), SEQ ID NO: 19 (IgG3 Fc domain), or SEQ ID NO: 21 (IgG4 Fc domain).
  • Embodiment 12 the circular polyribonucleotide of embodiment 11, wherein the IgG4 Fc domain comprises an amino acid sequence of SEQ ID NO: 21.
  • Embodiment 13 is the circular polyribonucleotide of any one of embodiments 8-12, wherein the sequence encoding the polypeptide comprises a nucleic acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16 (IgG1 Fc domain), SEQ ID NO: 18 (IgG2 Fc domain), SEQ ID NO: 20 (IgG3 Fc domain), or SEQ ID NO: 22 (IgG4 Fc domain).
  • Embodiment 14 is the circular polyribonucleotide of embodiment 13, wherein the sequence encoding the polypeptide comprises a nucleic acid sequence of SEQ ID NO: 16 (IgG1 Fc domain), SEQ ID NO: 18 (IgG2 Fc domain), SEQ ID NO: 20 (IgG3 Fc domain), or SEQ ID NO: 22 (IgG4 Fc domain).
  • Embodiment 15 is the circular polyribonucleotide of any one of embodiments 8-14, wherein the Fc domain comprises a mutation.
  • Embodiment 16 is the circular polyribonucleotide of any one of embodiments 8-10, 13, and 15, wherein the Fc domain comprises the amino acid sequence of SEQ ID NO: 15 (IgG1 Fc domain), SEQ ID NO: 17 (IgG2 Fc domain), SEQ ID NO: 19 (IgG3 Fc domain), or SEQ ID NO: 21 (IgG4 Fc domain), except the amino acid sequence of SEQ ID NO: 15 (IgG1 Fc domain), SEQ ID NO: 17 (IgG2 Fc domain), SEQ ID NO: 19 (IgG3 Fc domain), or SEQ ID NO: 21 (IgG4 Fc domain) comprises an amino acid substitution selected from one or more of: M252Y/S254T/T256E (YTE); L284A/L285A; M428L/N434S (LS); T307A/E380A/N434A (AAA); T250Q/M428L (QL), and V308P.
  • Embodiment 17 is the circular polyribonucleotide of embodiment 8, wherein the albumin polypeptide comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23.
  • Embodiment 18 is the circular polyribonucleotide of embodiment 8, wherein the peptide moiety comprises a nanobody that binds to albumin.
  • Embodiment 19 is the circular polyribonucleotide of any one of embodiments 1-18, wherein the circular polyribonucleotide comprises one or more modifications.
  • Embodiment 20 is the circular polyribonucleotide of embodiment 19, wherein the one or more modifications comprise one or more modifications to a portion of the sequence encoding the GLP-2 polypeptide or analog thereof.
  • Embodiment 21 is the circular polyribonucleotide of embodiment 19 or embodiment 20, wherein the one or more modifications comprise a modification to a sugar, a nucleobase, an internucleoside linkage, or a combination thereof.
  • Embodiment 22 is the circular polyribonucleotide of any one of embodiments 1-21, wherein the circular polyribonucleotide further comprises a translation initiation sequence operably linked to the sequence encoding the polypeptide.
  • Embodiment 23 is the circular polyribonucleotide of any one of embodiments 1-22, wherein the circular polyribonucleotide further comprises an internal ribosome entry site (IRES) operatively linked to the sequence encoding the polypeptide.
  • IRS internal ribosome entry site
  • Embodiment 24 is the circular polyribonucleotide of any one of embodiments 1-23, wherein the circular polyribonucleotide further comprises a translation termination sequence.
  • Embodiment 2 is the circular polyribonucleotide of any one of embodiments 1-24, wherein the circular polyribonucleotide lacks a translation termination sequence.
  • Embodiment 26 is the circular polyribonucleotide of any one of embodiments 1-25, wherein the circular polyribonucleotide further comprises a stagger element at a 3’ end of the sequence encoding the polypeptide.
  • Embodiment 27 is the circular polyribonucleotide of embodiment 26, wherein the stagger element is configured to stall a ribosome during rolling circle translation.
  • Embodiment 28 is the circular polyribonucleotide of any one of embodiments 1-27, wherein the circular polyribonucleotide further comprises a replication element.
  • Embodiment 29 is the circular polyribonucleotide of any one of embodiments 1-28, wherein the circular polyribonucleotide further comprises a 5’ untranslated region operably linked to the sequence encoding the polypeptide.
  • Embodiment 30 is the circular polyribonucleotide of any one of embodiments 1-29, wherein the circular polyribonucleotide does not comprise a poly(A) sequence operably linked to the sequence encoding the polypeptide.
  • Embodiment 31 is the circular polyribonucleotide of any one of embodiments 1-30, wherein the circular polyribonucleotide does not comprise a 5’ untranslated region operably linked to the sequence encoding the polypeptide.
  • Embodiment 32 is the circular polyribonucleotide of any one of embodiments 1-31, wherein the secretion signal sequence is a liver-specific secretion signal sequence, wherein the LRN Ref. No.: LRN23-107WO Fish Ref.
  • No.: 56929-0005WO1 liver-specific secretion signal comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO
  • Embodiment 33 is the circular polyribonucleotide of any one of embodiments 1-31, wherein the secretion signal sequence comprises an IL-2 secretion signal sequence, a proglucagon secretion signal sequence, a Gaussia luciferase secretion signal sequence, or a erythropoietin (EPO) secretion signal sequence.
  • Embodiment 34 is the circular polyribonucleotide of embodiment 33, wherein the IL-2 secretion signal sequence comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 29.
  • Embodiment 35 is the circular polyribonucleotide of embodiment 33, wherein the proglucagon secretion signal sequence comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 31.
  • Embodiment 36 is the circular polyribonucleotide of embodiment 33, wherein the Gaussia luciferase secretion signal sequence comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 33.
  • Embodiment 37 is the circular polyribonucleotide of embodiment 33, wherein the EPO secretion signal sequence comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 35.
  • Embodiment 38 is the circular polyribonucleotide of embodiment 33, wherein the sequence encoding the polypeptide comprises a nucleic acid sequence that is at least 70%, 80%, LRN Ref. No.: LRN23-107WO Fish Ref. No.: 56929-0005WO1 90%, 95%, 99%, or 100% identical to one of SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39.
  • Embodiment 39 is the circular polyribonucleotide of any one of embodiments 1-38, wherein the circular polyribonucleotide further comprises at least one spacer sequence.
  • Embodiment 40 is the circular polyribonucleotide of embodiment 39, wherein the polypeptide comprises a protease cleavage site and the at least one spacer sequence encodes the protease cleavage site.
  • Embodiment 41 is the circular polyribonucleotide of embodiment 40, wherein the protease cleavage site is positioned between (a) the secretion signal and (b) the GLP-2 polypeptide or analog thereof and the half-life extension moiety.
  • Embodiment 42 is the circular polyribonucleotide of any one of embodiments 1-41, wherein the polypeptide further comprises at least one linker sequence.
  • Embodiment 43 is the circular polyribonucleotide of embodiment 42, wherein the at least one linker sequence encodes an amino acid sequence selected from (GS) x , (GGS) x , (GGGGS) x (SEQ ID NO: 141), (GGSG)x (SEQ ID NO: 142), and (SGGG)x (SEQ ID NO: 143), wherein x is an integer from 1 to 50.
  • Embodiment 44 is the circular polyribonucleotide of embodiment 42 or 43, wherein the at least one linker sequence is disposed between the GLP-2 polypeptide or analog thereof and the half-life extension moiety.
  • Embodiment 45 is the circular polyribonucleotide of any one of embodiments 1-44, wherein the circular polyribonucleotide comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.
  • Embodiment 46 is the circular polyribonucleotide of any one of embodiments 1-45, wherein the circular polyribonucleotide comprises an amino acid sequence of SEQ ID NO: 1.
  • Embodiment 47 is the circular polyribonucleotide of any one of embodiments 1-46, wherein the circular polyribonucleotide comprises a nucleic acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2.
  • Embodiment 48 is the circular polyribonucleotide of any one of embodiments 1-47, wherein the circular polyribonucleotide comprises a nucleic acid sequence of SEQ ID NO: 2.
  • Embodiment 49 is a pharmaceutical composition comprising the circular polyribonucleotide of any one of embodiments 1-48, and a pharmaceutically acceptable excipient.
  • Embodiment 50 is the pharmaceutical composition of embodiment 49, wherein the circular polyribonucleotide is formulated as a lipid nanoparticle.
  • Embodiment 51 is the pharmaceutical composition of embodiment 49, wherein the circular polyribonucleotide is formulated for unencapsulated delivery or natural particle delivery.
  • Embodiment 52 is the pharmaceutical composition of any one of embodiments 49-51, wherein the pharmaceutical composition is formulated for intravenous, subcutaneous, intrahepatic, or intramuscular administration.
  • Embodiment 53 is the pharmaceutical composition of any one of embodiments 49-51, wherein the pharmaceutical composition is formulated for local administration.
  • Embodiment 54 is the pharmaceutical composition of any one of embodiments 49-51, wherein the pharmaceutical composition is formulated for systemic administration.
  • Embodiment 55 is the pharmaceutical composition of any one of embodiments 49-54, wherein the pharmaceutical composition comprises one or more of the following features: (i) a concentration of the circular polyribonucleotide of between 0.1 ng/mL and 500 mg/mL; (ii) a concentration of deoxyribonucleotide of no more than 100 ng/mL; (iii) a concentration of protein of less than 100 ng of protein per milligram (mg) of the circular polyribonucleotide; (iv) an A260/A280 absorbance ratio of from about 1.6 to 2.3 as measured by a spectrophotometer; (v) substantially free of a process-related impurity selected from a cell protein, a
  • Embodiment 56 is a method of treating a glucagon-like peptide-2 (GLP-2)-related disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a circular polyribonucleotide of any one of embodiments 1-48 or the pharmaceutical composition of any one of embodiments 49-55.
  • Embodiment 57 is the method of embodiment 56, wherein the GLP-2-related disease is selected from the group consisting of: short bowel syndrome (SBS), short bowel syndrome with intestinal failure (SBS-IF), pediatric SBS, adult SBS, major small bowel resection, total LRN Ref. No.: LRN23-107WO Fish Ref.
  • SBS short bowel syndrome
  • SBS-IF short bowel syndrome with intestinal failure
  • pediatric SBS pediatric SBS
  • adult SBS major small bowel resection
  • Embodiment 58 is a method of increasing intestinal growth in a subject in need thereof, the method comprising administering to the subject an effective amount of a circular polyribonucleotide of any one of embodiments 1-48 or the pharmaceutical composition of any one of embodiments 49-55.
  • Embodiment 59 is the method of embodiment 59, wherein increasing intestinal growth comprises increasing crypt cell growth.
  • Embodiment 60 is a method of increasing intestinal absorption of fluids and nutrients in a subject in need thereof comprising administering to the subject an effective amount of a circular polyribonucleotide of any one of embodiments 1-48 or the pharmaceutical composition of any one of embodiments 49-55.
  • Embodiment 61 is the method of any one of embodiments 58-60, wherein the subject has short bowel syndrome (SBS), short bowel syndrome with intestinal failure (SBS-IF), pediatric SBS, adult SBS, major small bowel resection, total parenteral nutrition (TPN)-induced intestinal hypoplasia, small and large intestinal inflammation, Crohn’s disease, chemotherapy-induced mucositis, and intestinal ischemia–reperfusion injury.
  • SBS short bowel syndrome
  • SBS-IF short bowel syndrome with intestinal failure
  • TPN total parenteral nutrition
  • Embodiment 62 is the method of any one of embodiments 56-61, wherein the circular polyribonucleotide of embodiments 1-48 or the pharmaceutical composition of any one of embodiments 49-55 is administered one or more times to the subject.
  • Embodiment 63 is the method of any one of embodiments 56-62, wherein the circular polyribonucleotide of embodiments 1-48 or the pharmaceutical composition of any one of embodiments 49-55 is administered locally.
  • Embodiment 64 is the method of any one of embodiments 56-62, wherein the circular polyribonucleotide of embodiments 1-48 or the pharmaceutical composition of any one of embodiments 49-55 is administered systemically.
  • Embodiment 65 is the method of any one of embodiments 56-64, further comprising administering one or more additional therapeutic agents to the subject.
  • Embodiment 66 is the method of any one of embodiments 56-65, wherein the subject is a human subject.
  • Embodiment 67 is a kit comprising: (a) (i) a composition comprising the circular polyribonucleotide of any one of embodiments 1-48 or (ii) the pharmaceutical composition of any one of embodiments 49-55; and (b) instructions for administering the composition or the pharmaceutical composition to a subject in need thereof.
  • Embodiment 68 is a circular polyribonucleotide comprising a sequence encoding a polypeptide comprising (i) a glucagon-like peptide-2 (GLP-2) polypeptide or analog thereof, and (ii) a half-life extension moiety.
  • Embodiment 69 is a circular polyribonucleotide comprising a sequence encoding a polypeptide comprising (i) a glucagon-like peptide-2 (GLP-2) polypeptide or analog thereof, and (ii) a half-life extension moiety, wherein the half-life extension moiety is an Fc domain comprising a M252Y/S254T/T256E (YTE) mutation.
  • Embodiment 70 is a modified linear RNA comprising a sequence encoding a polypeptide comprising a glucagon-like peptide-2 (GLP-2) or analog thereof.
  • Embodiment 71 is a modified linear RNA comprising a sequence encoding a polypeptide comprising (i) a glucagon-like peptide-2 (GLP-2) polypeptide or analog thereof, and (ii) a secretion signal sequence.
  • Embodiment 72 is the modified linear RNA of embodiment 70 or 71, further comprising a half-life extension moiety.

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Abstract

La présente divulgation concerne des polyribonucléotides circulaires comprenant une séquence codant un polypeptide comprenant (i) un polypeptide de peptide-2 de type glucagon (GLP-2) ou un analogue de celui-ci, (ii) une fraction d'extension de demi-vie, et (iii) une séquence de signaux de sécrétion, ainsi que des kits et des compositions de ceux-ci. La présente divulgation concerne également des méthodes de traitement de maladies associées à GLP-2 avec des compositions présentement décrites.
PCT/US2024/056294 2023-11-17 2024-11-15 Polyribonucléotides circulaires codant le peptide 2 de type glucagon (glp-2) et leurs utilisations Pending WO2025106930A1 (fr)

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